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1.
Molecules ; 28(6)2023 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-36985425

RESUMO

The naphthalene diimide compound QN-302, designed to bind to G-quadruplex DNA sequences within the promoter regions of cancer-related genes, has high anti-proliferative activity in pancreatic cancer cell lines and anti-tumor activity in several experimental models for the disease. We show here that QN-302 also causes downregulation of the expression of the S100P gene and the S100P protein in cells and in vivo. This protein is well established as being involved in key proliferation and motility pathways in several human cancers and has been identified as a potential biomarker in pancreatic cancer. The S100P gene contains 60 putative quadruplex-forming sequences, one of which is in the promoter region, 48 nucleotides upstream from the transcription start site. We report biophysical and molecular modeling studies showing that this sequence forms a highly stable G-quadruplex in vitro, which is further stabilized by QN-302. We also report transcriptome analyses showing that S100P expression is highly upregulated in tissues from human pancreatic cancer tumors, compared to normal pancreas material. The extent of upregulation is dependent on the degree of differentiation of tumor cells, with the most poorly differentiated, from more advanced disease, having the highest level of S100P expression. The experimental drug QN-302 is currently in pre-IND development (as of Q1 2023), and its ability to downregulate S100P protein expression supports a role for this protein as a marker of therapeutic response in pancreatic cancer. These results are also consistent with the hypothesis that the S100P promoter G-quadruplex is a potential therapeutic target in pancreatic cancer at the transcriptional level for QN-302.


Assuntos
Quadruplex G , Neoplasias Pancreáticas , Humanos , Linhagem Celular Tumoral , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Expressão Gênica , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas
2.
Br J Cancer ; 122(6): 847-856, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31937926

RESUMO

BACKGROUND: Ovarian cancer has a poor survival rate due to late diagnosis and improved methods are needed for its early detection. Our primary objective was to identify and incorporate additional biomarkers into longitudinal models to improve on the performance of CA125 as a first-line screening test for ovarian cancer. METHODS: This case-control study nested within UKCTOCS used 490 serial serum samples from 49 women later diagnosed with ovarian cancer and 31 control women who were cancer-free. Proteomics-based biomarker discovery was carried out using pooled samples and selected candidates, including those from the literature, assayed in all serial samples. Multimarker longitudinal models were derived and tested against CA125 for early detection of ovarian cancer. RESULTS: The best performing models, incorporating CA125, HE4, CHI3L1, PEBP4 and/or AGR2, provided 85.7% sensitivity at 95.4% specificity up to 1 year before diagnosis, significantly improving on CA125 alone. For Type II cases (mostly high-grade serous), models achieved 95.5% sensitivity at 95.4% specificity. Predictive values were elevated earlier than CA125, showing the potential of models to improve lead time. CONCLUSIONS: We have identified candidate biomarkers and tested longitudinal multimarker models that significantly improve on CA125 for early detection of ovarian cancer. These models now warrant independent validation.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Ovarianas/diagnóstico , Proteômica/métodos , Idoso , Estudos de Casos e Controles , Detecção Precoce de Câncer , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Taxa de Sobrevida
3.
Clin Proteomics ; 16: 14, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30992697

RESUMO

BACKGROUND: Endometriosis is a common gynaecological disorder affecting 5-10% of women of reproductive age who often experience chronic pelvic pain and infertility. Definitive diagnosis is through laparoscopy, exposing patients to potentially serious complications, and is often delayed. Non-invasive biomarkers are urgently required to accelerate diagnosis and for triaging potential patients for surgery. METHODS: This retrospective case control biomarker discovery and validation study used quantitative 2D-difference gel electrophoresis and tandem mass tagging-liquid chromatography-tandem mass spectrometry for protein expression profiling of eutopic and ectopic endometrial tissue samples collected from 28 cases of endometriosis and 18 control patients undergoing surgery for investigation of chronic pelvic pain without endometriosis or prophylactic surgery. Samples were further sub-grouped by menstrual cycle phase. Selected differentially expressed candidate markers (LUM, CPM, TNC, TPM2 and PAEP) were verified by ELISA in a set of 87 serum samples collected from the same and additional women. Previously reported biomarkers (CA125, sICAM1, FST, VEGF, MCP1, MIF and IL1R2) were also validated and diagnostic performance of markers and combinations established. RESULTS: Cycle phase and endometriosis-associated proteomic changes were identified in eutopic tissue from over 1400 identified gene products, yielding potential biomarker candidates. Bioinformatics analysis revealed enrichment of adhesion/extracellular matrix proteins and progesterone signalling. The best single marker for discriminating endometriosis from controls remained CA125 (AUC = 0.63), with the best cross-validated multimarker models improving the AUC to 0.71-0.81, depending upon menstrual cycle phase and control group. CONCLUSIONS: We have identified menstrual cycle- and endometriosis-associated protein changes linked to various cellular processes that are potential biomarkers and that provide insight into the biology of endometriosis. Our data indicate that the markers tested, whilst not useful alone, have improved diagnostic accuracy when used in combination and demonstrate menstrual cycle specificity. Tissue heterogeneity and blood contamination is likely to have hindered biomarker discovery, whilst a small sample size precludes accurate determination of performance by cycle phase. Independent validation of these biomarker panels in a larger cohort is however warranted, and if successful, they may have clinical utility in triaging patients for surgery.

4.
Mol Cell Proteomics ; 16(4): 608-621, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28174229

RESUMO

Most breast cancers arise from luminal epithelial cells, and 25-30% of these tumors overexpress the ErbB2/HER2 receptor that correlates with disease progression and poor prognosis. The mechanisms of ErbB2 signaling and the effects of its overexpression are not fully understood. Herein, stable isotope labeling by amino acids in cell culture (SILAC), expression profiling, and phosphopeptide enrichment of a relevant, non-transformed, and immortalized human mammary luminal epithelial cell model were used to profile ErbB2-dependent differences in protein expression and phosphorylation events triggered via EGF receptor (EGF treatment) and ErbB3 (HRG1ß treatment) in the context of ErbB2 overexpression. Bioinformatics analysis was used to infer changes in cellular processes and signaling events. We demonstrate the complexity of the responses to oncogene expression and growth factor signaling, and we identify protein changes relevant to ErbB2-dependent altered cellular phenotype, in particular cell cycle progression and hyper-proliferation, reduced adhesion, and enhanced motility. Moreover, we define a novel mechanism by which ErbB signaling suppresses basal interferon signaling that would promote the survival and proliferation of mammary luminal epithelial cells. Numerous novel sites of growth factor-regulated phosphorylation were identified that were enhanced by ErbB2 overexpression, and we putatively link these to altered cell behavior and also highlight the importance of performing parallel protein expression profiling alongside phosphoproteomic analysis.


Assuntos
Células Epiteliais/citologia , Perfilação da Expressão Gênica/métodos , Glândulas Mamárias Humanas/metabolismo , Fosfoproteínas/metabolismo , Proteômica/métodos , Receptor ErbB-2/genética , Adesão Celular , Técnicas de Cultura de Células , Linhagem Celular , Movimento Celular , Proliferação de Células , Biologia Computacional/métodos , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/metabolismo , Feminino , Amplificação de Genes , Humanos , Marcação por Isótopo , Ligantes , Glândulas Mamárias Humanas/citologia , Neuregulina-1/farmacologia , Mapas de Interação de Proteínas , Receptor ErbB-2/metabolismo , Transdução de Sinais , Regulação para Cima
5.
Prostate ; 77(15): 1539-1547, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28944496

RESUMO

BACKGROUND: OCT1002 is a unidirectional hypoxia-activated prodrug (uHAP) OCT1002 that can target hypoxic tumor cells. Hypoxia is a common feature in prostate tumors and is known to drive disease progression and metastasis. It is, therefore, a rational therapeutic strategy to directly target hypoxic tumor cells in an attempt to improve treatment for this disease. Here we tested OCT1002 alone and in combination with standard-of-care agents in hypoxic models of castrate-resistant prostate cancer (CRPC). METHODS: The effect of OCT1002 on tumor growth and vasculature was measured using murine PC3 xenograft and dorsal skin fold (DSF) window chamber models. The effects of abiraterone, docetaxel, and cabazitaxel, both singly and in combination with OCT1002, were also compared. RESULTS: The hypoxia-targeting ability of OCT1002 effectively controls PC3 tumor growth. The effect was evident for at least 42 days after exposure to a single dose (30 mg/kg) and was comparable to, or better than, drugs currently used in the clinic. In DSF experiments OCT1002 caused vascular collapse in the PC3 tumors and inhibited the revascularization seen in controls. In this model OCT1002 also enhanced the anti-tumor effects of abiraterone, cabazitaxel, and docetaxel; an effect which was accompanied by a more prolonged reduction in tumor vasculature density. CONCLUSIONS: These studies provide the first evidence that OCT1002 can be an effective agent in treating hypoxic, castrate-resistant prostate tumors, either singly or in combination with established chemotherapeutics for prostate cancer.


Assuntos
Antraquinonas/farmacologia , Etilenodiaminas/farmacologia , Pró-Fármacos/farmacologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Animais , Antraquinonas/farmacocinética , Processos de Crescimento Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Etilenodiaminas/farmacocinética , Humanos , Masculino , Camundongos , Camundongos Nus , Pró-Fármacos/farmacocinética , Neoplasias de Próstata Resistentes à Castração/irrigação sanguínea , Neoplasias de Próstata Resistentes à Castração/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
6.
J Cell Physiol ; 231(2): 473-82, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26189652

RESUMO

Aberrant expression of the transcription factor RUNX2 in prostate cancer has a number of important consequences including increased resistance to apoptosis, invasion and metastasis to bone. We previously demonstrated that hypoxia up-regulated RUNX2 in tumour cells, which in turn up-regulated the anti-apoptotic factor Bcl-2. Here, we investigate the impact of nitric oxide (NO) on RUNX2 and Bcl-2 expression in prostate cancer and further, how RUNX2 over-expression can impact tumour growth, angiogenesis and oxygenation in vivo. The effect of NO levels on RUNX2 and thus Bcl-2 expression was examined in prostate cancer cells in vitro using methods including gene and protein expression analyses, nitrite quantitation, protein-DNA interaction assays (ChIP) and viability assays (XTT). The effect of RUNX2 over-expression on tumour physiology (growth, oxygenation and angiogenesis) was also assessed in vivo using LNCaP xenografts. A low (but not high) concentration of NO (10 µM) induced expression of RUNX2 and Bcl-2, conferring resistance to docetaxel. These effects were induced via the ERK and PI3K pathways and were dependent on intact AP-1 binding sites in the RUNX2 promoter. RUNX2 over-expression in LNCaP tumours in vivo decreased the time to tumour presentation and increased tumour growth. Moreover, these tumours exhibited improved tumour angiogenesis and oxygenation. Low levels of NO increase expression of RUNX2 and Bcl-2 in LNCaP prostate tumour cells, and in vivo up-regulation of RUNX2 created tumours with a more malignant phenotype. Collectively, our data reveals the importance of NO-regulation of key factors in prostate cancer disease progression.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Óxido Nítrico/metabolismo , Neoplasias da Próstata/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Subunidade alfa 1 de Fator de Ligação ao Core/antagonistas & inibidores , Feminino , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , RNA Interferente Pequeno/genética , Regulação para Cima
7.
Int J Cancer ; 137(8): 1806-17, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25204737

RESUMO

Epithelial ovarian cancer (EOC) is still considered the most lethal gynecological malignancy and improved early detection of ovarian cancer is crucial to improving patient prognoses. To address this need, we tested whether candidate EOC biomarkers can be identified using three-dimensional (3D) in vitro models. We quantified changes in the abundance of secreted proteins in a 3D genetic model of early-stage EOC, generated by expressing CMYC and KRAS(G) (12) (V) in TERT-immortalized normal ovarian epithelial cells. Cellular proteins were labeled in live cells using stable isotopic amino acid analogues, and secreted proteins identified and quantified using liquid chromatography-tandem mass spectrometry. Thirty-seven and 55 proteins were differentially expressed by CMYC and CMYC+KRAS(G) (12) (V) expressing cells respectively (p < 0.05; >2-fold). We evaluated expression of the top candidate biomarkers in ∼210 primary EOCs: CHI3L1 and FKBP4 are both expressed by >96% of primary EOCs, and FASN and API5 are expressed by 86 and 75% of cases. High expression of CHI3L1 and FKBP4 was associated with worse patient survival (p = 0.042 and p = 0.002, respectively). Expression of LGALS3BP was positively associated with recurrence (p = 0.0001) and suboptimal debulking (p = 0.018) suggesting that these proteins may be novel prognostic biomarkers. Furthermore, within early stage tumours (I/II), high expression of API5, CHI3L1 and FASN was associated with high tumour grade (p = 3 × 10(-4) , p = 0.016, p = 0.010, respectively). We show in vitro cell biology models of early-stage cancer development can be used to identify novel candidate biomarkers for disease, and report the identification of proteins that represent novel potential candidate diagnostic and prognostic biomarkers for this highly lethal disease.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Adipocinas/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Proteína 1 Semelhante à Quitinase-3 , Ácido Graxo Sintase Tipo I/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas In Vitro , Lectinas/metabolismo , Modelos Genéticos , Neoplasias Epiteliais e Glandulares/patologia , Proteínas Nucleares/metabolismo , Neoplasias Ovarianas/patologia , Prognóstico , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Proteínas ras/metabolismo
8.
Int J Cancer ; 132(6): 1323-32, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22915157

RESUMO

Androgen withdrawal induces hypoxia in androgen-sensitive tissue; this is important as in the tumour microenvironment, hypoxia is known to drive malignant progression. Our study examined the time-dependent effect of androgen deprivation therapy (ADT) on tumour oxygenation and investigated the role of ADT-induced hypoxia on malignant progression in prostate tumours. LNCaP xenografted tumours were treated with anti-androgens and tumour oxygenation measured. Dorsal skin fold (DSF) chambers were used to image tumour vasculature in vivo. Quantitative PCR (QPCR) identified differential gene expression following treatment with bicalutamide. Bicalutamide-treated and vehicle-only-treated tumours were re-established in vitro, and invasion and sensitivity to docetaxel were measured. Tumour growth delay was calculated following treatment with bicalutamide combined with the bioreductive drug AQ4N. Tumour oxygenation measurements showed a precipitate decrease following initiation of ADT. A clinically relevant dose of bicalutamide (2 mg/kg/day) decreased tumour oxygenation by 45% within 24 hr, reaching a nadir of 0.09% oxygen (0.67 ± 0.06 mmHg) by Day 7; this persisted until Day 14 when it increased up to Day 28. Using DSF chambers, LNCaP tumours treated with bicalutamide showed loss of small vessels at Days 7 and 14 with revascularisation occurring by Day 21. QPCR showed changes in gene expression consistent with the vascular changes and malignant progression. Cells from bicalutamide-treated tumours were more malignant than vehicle-treated controls. Combining bicalutamide with AQ4N (50 mg/kg, single dose) caused greater tumour growth delay than bicalutamide alone. Our study shows that bicalutamide-induced hypoxia selects for cells that show malignant progression; targeting hypoxic cells may provide greater clinical benefit.


Assuntos
Antagonistas de Androgênios/farmacologia , Anilidas/farmacologia , Antraquinonas/administração & dosagem , Hipóxia Celular , Nitrilas/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Compostos de Tosil/farmacologia , Animais , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/metabolismo , Transdução de Sinais , Fatores de Tempo
9.
Antibodies (Basel) ; 12(4)2023 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-37873862

RESUMO

Antibody-drug conjugates (ADCs) constitute a rapidly expanding category of biopharmaceuticals that are reshaping the landscape of targeted chemotherapy. The meticulous process of selecting therapeutic targets, aided by specific monoclonal antibodies' high specificity for binding to designated antigenic epitopes, is pivotal in ADC research and development. Despite ADCs' intrinsic ability to differentiate between healthy and cancerous cells, developmental challenges persist. In this study, we present a rationalized pipeline encompassing the initial phases of the ADC development, including target identification and validation. Leveraging an in-house, computationally constructed ADC target database, termed ADC Target Vault, we identified a set of novel ovarian cancer targets. We effectively demonstrate the efficacy of Surface Plasmon Resonance (SPR) technology and in vitro models as predictive tools, expediting the selection and validation of targets as ADC candidates for ovarian cancer therapy. Our analysis reveals three novel robust antibody/target pairs with strong binding and favourable antibody internalization rates in both wild-type and cisplatin-resistant ovarian cancer cell lines. This approach enhances ADC development and offers a comprehensive method for assessing target/antibody combinations and pre-payload conjugation biological activity. Additionally, the strategy establishes a robust platform for high-throughput screening of potential ovarian cancer ADC targets, an approach that is equally applicable to other cancer types.

10.
Cancer Discov ; 13(3): 672-701, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36745048

RESUMO

Drugs that kill tumors through multiple mechanisms have the potential for broad clinical benefits. Here, we first developed an in silico multiomics approach (BipotentR) to find cancer cell-specific regulators that simultaneously modulate tumor immunity and another oncogenic pathway and then used it to identify 38 candidate immune-metabolic regulators. We show the tumor activities of these regulators stratify patients with melanoma by their response to anti-PD-1 using machine learning and deep neural approaches, which improve the predictive power of current biomarkers. The topmost identified regulator, ESRRA, is activated in immunotherapy-resistant tumors. Its inhibition killed tumors by suppressing energy metabolism and activating two immune mechanisms: (i) cytokine induction, causing proinflammatory macrophage polarization, and (ii) antigen-presentation stimulation, recruiting CD8+ T cells into tumors. We also demonstrate a wide utility of BipotentR by applying it to angiogenesis and growth suppressor evasion pathways. BipotentR (http://bipotentr.dfci.harvard.edu/) provides a resource for evaluating patient response and discovering drug targets that act simultaneously through multiple mechanisms. SIGNIFICANCE: BipotentR presents resources for evaluating patient response and identifying targets for drugs that can kill tumors through multiple mechanisms concurrently. Inhibition of the topmost candidate target killed tumors by suppressing energy metabolism and effects on two immune mechanisms. This article is highlighted in the In This Issue feature, p. 517.


Assuntos
Antineoplásicos , Melanoma , Humanos , Antineoplásicos/farmacologia , Receptores de Estrogênio , Imunoterapia , Melanoma/patologia , Linfócitos T CD8-Positivos , Microambiente Tumoral , Linhagem Celular Tumoral , Receptor ERRalfa Relacionado ao Estrogênio
11.
Front Oncol ; 12: 1014280, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36505806

RESUMO

Background: Ovarian cancer (OC) is amongst the most lethal of common cancers in women. Lacking in specific symptoms in the early stages, OC is predominantly diagnosed late when the disease has undergone metastatic spread and chemotherapy is relied on to prolong life. Platinum-based therapies are preferred and although many tumors respond initially, the emergence of platinum-resistance occurs in the majority of cases after which prognosis is very poor. Upregulation of DNA damage pathways is a common feature of platinum resistance in OC with cyclin dependent kinases (CDKs) serving as key regulators of this process and suggesting that CDK inhibitors (CDKis) could be effective tools in the treatment of platinum resistant and refractory OC. Aim: The aim of this study was to evaluate the efficacy of CDKis in platinum resistant OC models and serve as a predictor of potential clinical utility. Methods: The efficacy of CDKi, dinaciclib, was determined in wildtype and platinum resistant cell line pairs representing different OC subtypes. In addition, dinaciclib was evaluated in primary cells isolated from platinum-sensitive and platinum-refractory tumors to increase the clinical relevance of the study. Results and conclusions: Dinaciclib proved highly efficacious in OC cell lines and primary cells, which were over a thousand-fold more sensitive to the CDKi than to cisplatin. Furthermore, cisplatin resistance in these cells did not influence sensitivity to dinaciclib and the two drugs combined additively in both platinum-sensitive and platinum-resistant OC cells suggesting a potential role for pan-CDKis (CDKis targeting multiple CDKs), such as dinaciclib, in the treatment of advanced and platinum-resistant OC.

12.
Mol Cancer Res ; 20(6): 841-853, 2022 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-35302608

RESUMO

Inhibiting androgen signaling using androgen signaling inhibitors (ASI) remains the primary treatment for castrate-resistant prostate cancer. Acquired resistance to androgen receptor (AR)-targeted therapy represents a major impediment to durable clinical response. Understanding resistance mechanisms, including the role of AR expressed in other cell types within the tumor microenvironment, will extend the clinical benefit of AR-targeted therapy. Here, we show the ASI enzalutamide induces vascular catastrophe and promotes hypoxia and microenvironment adaptation. We characterize treatment-induced hypoxia, and subsequent induction of angiogenesis, as novel mechanisms of relapse to enzalutamide, highlighting the importance of two hypoxia-regulated cytokines in underpinning relapse. We confirmed AR expression in CD34+ vascular endothelium of biopsy tissue and human vascular endothelial cells (HVEC). Enzalutamide attenuated angiogenic tubule formation and induced cytotoxicity in HVECs in vitro, and rapidly induced sustained hypoxia in LNCaP xenografts. Subsequent reoxygenation, following prolonged enzalutamide treatment, was associated with increased tumor vessel density and accelerated tumor growth. Hypoxia increased AR expression and transcriptional activity in prostate cells in vitro. Coinhibition of IL8 and VEGF-A restored tumor response in the presence of enzalutamide, confirming the functional importance of their elevated expression in enzalutamide-resistant models. Moreover, coinhibition of IL8 and VEGF-A resulted in a durable, effective resolution of enzalutamide-sensitive prostate tumors. We conclude that concurrent inhibition of two hypoxia-induced factors, IL8 and VEGF-A, prolongs tumor sensitivity to enzalutamide in preclinical models and may delay the onset of enzalutamide resistance. IMPLICATIONS: Targeting hypoxia-induced signaling may extend the therapeutic benefit of enzalutamide, providing an improved treatment strategy for patients with resistant disease.


Assuntos
Antagonistas de Receptores de Andrógenos , Neoplasias de Próstata Resistentes à Castração , Antagonistas de Androgênios/farmacologia , Antagonistas de Receptores de Andrógenos/farmacologia , Androgênios/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Células Endoteliais/metabolismo , Humanos , Hipóxia/tratamento farmacológico , Interleucina-8/genética , Masculino , Recidiva Local de Neoplasia/tratamento farmacológico , Nitrilas/farmacologia , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/genética
13.
Proteomics ; 11(23): 4583-7, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21932442

RESUMO

Protein regulation by reversible phosphorylation is fundamental in nature, and large-scale phosphoproteomic analyses are becoming routine in proteomics laboratories. These analyses utilise phosphopeptide separation and enrichment techniques linked to LC-MS/MS. Herein, we report that IMAC and TiO(2) also enrich for non-phosphorylated modified peptides such as acetylated, deamidated and carbamylated peptides. Urea and digestion conditions commonly used in phosphoproteomic workflows are the likely sources of the induced modifications (deamidation and carbamylation) and can easily modify phosphopeptides. Including these variable modifications in database searches increased the total number of identified phosphopeptides by 15%. We also show that strong cation exchange fractionation provides poor resolution of phosphopeptides and actually enriches these alternatively modified peptides. By switching to reverse-phase chromatography, we show a significant improvement in the number of identified phosphopeptides. We recommend that the users of phosphopeptide enrichment strategies avoid using urea as a denaturant and that careful consideration is given to chromatographic conditions and the types of variable modifications used in database searches. Thus, the capacity of IMAC and TiO(2) to enrich phosphopeptides bearing modifications other than phosphorylation is a previously unappreciated property of these chromatographies with practical implications for the field of phosphoproteomics.


Assuntos
Cromatografia de Afinidade/métodos , Metais/química , Fosfopeptídeos/isolamento & purificação , Proteômica/métodos , Titânio/química , Bases de Dados de Proteínas
14.
Cancer Discov ; 11(5): 1118-1137, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33431496

RESUMO

Resistance to androgen receptor (AR) blockade in castration-resistant prostate cancer (CRPC) is associated with sustained AR signaling, including through alternative splicing of AR (AR-SV). Inhibitors of transcriptional coactivators that regulate AR activity, including the paralog histone acetyltransferase proteins p300 and CBP, are attractive therapeutic targets for lethal prostate cancer. Herein, we validate targeting p300/CBP as a therapeutic strategy for lethal prostate cancer and describe CCS1477, a novel small-molecule inhibitor of the p300/CBP conserved bromodomain. We show that CCS1477 inhibits cell proliferation in prostate cancer cell lines and decreases AR- and C-MYC-regulated gene expression. In AR-SV-driven models, CCS1477 has antitumor activity, regulating AR and C-MYC signaling. Early clinical studies suggest that CCS1477 modulates KLK3 blood levels and regulates CRPC biopsy biomarker expression. Overall, CCS1477 shows promise for the treatment of patients with advanced prostate cancer. SIGNIFICANCE: Treating CRPC remains challenging due to persistent AR signaling. Inhibiting transcriptional AR coactivators is an attractive therapeutic strategy. CCS1477, an inhibitor of p300/CBP, inhibits growth and AR activity in CRPC models, and can affect metastatic CRPC target expression in serial clinical biopsies.See related commentary by Rasool et al., p. 1011.This article is highlighted in the In This Issue feature, p. 995.


Assuntos
Antagonistas de Receptores de Andrógenos/uso terapêutico , Imidazóis/uso terapêutico , Oxazóis/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Fatores de Transcrição de p300-CBP/antagonistas & inibidores , Antagonistas de Receptores de Andrógenos/farmacologia , Animais , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Masculino , Camundongos , Oxazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
15.
J Gene Med ; 12(9): 755-65, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20821746

RESUMO

BACKGROUND: Using the tumour type specific human osteocalcin (hOC) promoter, we have previously reported strong promoter activation in hormone independent prostate cancer cells in vitro. In the present study, we present a comparative study of the tissue specific promoter prostate specific membrane antigen (PSMA), and the tumour-type specific hOC promoter driving the inducible nitric oxide synthase (iNOS) transgene using both in vitro and in vivo models. METHODS: In vitro cytotoxicity was assessed by clonogenic assay. Quantification of nitric oxide expression was determined by the Griess test. In vivo anti-tumour efficacy was determined by tumour growth delay following direct intra-tumoural injection of the constructs into PC3 xenografts. In addition, tumours were dissected post mortem and examined for morphological differences as well as changes in apoptotic protein expression. RESULTS: PSMA/iNOS produced cytotoxicity in both androgen dependant and independent cell lines. Nitric oxide quantification confirmed that increased cytotoxicity was directly associated with nitric oxide production. Tumour growth delays were observed in all groups treated with the iNOS-expressing constructs ranging from 10.7 days for the hOC/iNOS single dose treatment group to a maximum of 52.2 days for the hOC/iNOS multiple dose group. Intra-tumoural assessment of iNOS and cleaved poly (ADP-ribose) polymerase protein expression demonstrated a significant up-regulation of both proteins, indicating cytotoxicity mediated through the intrinsic apoptotic pathway. CONCLUSIONS: Highly significant tumour growth delay coupled with no detrimental side-effects were observed following treatment with the PSMA/iNOS and hOC/iNOS constructs. We consider that these findings provide a basis for the development of systemically delivered PSMA/iNOS or hOC/iNOS targeting early stage and advanced prostate cancer.


Assuntos
Regulação Neoplásica da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Terapia Genética , Neoplasias Hormônio-Dependentes/terapia , Óxido Nítrico Sintase Tipo II/genética , Neoplasias da Próstata/terapia , Animais , Antígenos de Superfície/genética , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Feminino , Glutamato Carboxipeptidase II/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Estadiamento de Neoplasias , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/patologia , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Osteocalcina/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Transgenes/fisiologia , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de Xenoenxerto
16.
BMC Cancer ; 10: 490, 2010 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-20840765

RESUMO

BACKGROUND: Members of the ErbB family of growth factor receptors are intricately linked with epithelial cell biology, development and tumourigenesis; however, the mechanisms involved in their downstream signalling are poorly understood. Indeed, it is unclear how signal specificity is achieved and the relative contribution each receptor has to specific gene expression. METHODS: Gene expression profiling of a human mammary luminal epithelial cell model of ErbB2-overexpression was carried out using cDNA microarrays with a common RNA reference approach to examine long-term overlapping and differential responses to EGF and heregulin beta1 treatment in the context of ErbB2 overexpression. Altered gene expression was validated using quantitative real time PCR and/or immunoblotting. One gene of interest was targeted for further characterisation, where the effects of siRNA-mediated silencing on IGF1-dependent signalling and cellular phenotype were examined and compared to the effects of loss of ErbB2 expression. RESULTS: 775 genes were differentially expressed and clustered in terms of their growth factor responsiveness. As well as identifying uncharacterized genes as novel targets of ErbB2-dependent signalling, ErbB2 overexpression augmented the induction of multiple genes involved in proliferation (e.g. MYC, MAP2K1, MAP2K3), autocrine growth factor signalling (VEGF, PDGF) and adhesion/cytoskeletal regulation (ZYX, THBS1, VCL, CNN3, ITGA2, ITGA3, NEDD9, TAGLN), linking them to the hyper-poliferative and altered adhesive phenotype of the ErbB2-overexpressing cells. We also report ErbB2-dependent down-regulation of multiple interferon-stimulated genes that may permit ErbB2-overexpressing cells to resist the anti-proliferative action of interferons. Finally, IGFBP3 was unique in its pattern of regulation and we further investigated a possible role for IGFBP3 down-regulation in ErbB2-dependent transformation through suppressed IGF1 signalling. We show that IGF1-dependent signalling and proliferation were enhanced in ErbB2-overexpressing cells, whilst loss of ErbB2 expression by siRNA silencing reduced IGF1 signalling. Furthermore, IGFBP3 knockdown resulted in basal ERK and Akt activation in luminal epithelial cells and increased invasiveness and anchorage-independent colony formation in SKBR3 cells. CONCLUSIONS: These data show IGFBP3 as a negative regulator of transformation and that its down-regulation enhances IGF1-dependent signalling. They also show that ErbB2 can up-regulate IGF1-dependent signalling, possibly via the regulated expression of IGFBP3.


Assuntos
Mama/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Fator de Crescimento Insulin-Like I/farmacologia , Receptor ErbB-2/metabolismo , Transdução de Sinais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Mama/patologia , Adesão Celular , Movimento Celular , Proliferação de Células , Fator de Crescimento Epidérmico/farmacologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Neuregulina-1/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
ACS Med Chem Lett ; 11(8): 1634-1644, 2020 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-32832034

RESUMO

Targeting of genomic quadruplexes is an approach to treating complex human cancers. We describe a series of tetra-substituted naphthalene diimide (ND) derivatives with a phenyl substituent directly attached to the ND core. The lead compound (SOP1812) has 10 times superior cellular and in vivo activity compared with previous ND compounds and nanomolar binding to human quadruplexes. The pharmacological properties of SOP1812 indicate good bioavailability, which is consistent with the in vivo activity in xenograft and genetic models for pancreatic cancer. Transcriptome analysis shows that it down-regulates several cancer gene pathways, including Wnt/ß-catenin signaling.

18.
J Gene Med ; 11(2): 160-8, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19062185

RESUMO

BACKGROUND: Nitric oxide (NO.) derived from donor drugs has been shown to be an effective chemosensitizer in vitro. We investigated the combination of inducible nitric oxide synthase (iNOS) gene transfer, driven by a strong constitutive promoter (cytomegalovirus; CMV) with the DNA cross-linking agent cisplatin in mouse and human tumour cell lines. METHODS: Proof of principal experiments were performed in the radiation-induced fibrosarcoma-1 (RIF-1) murine cell line. Cells were transfected with constitutively expressed CMV/iNOS plasmid DNA using a cationic lipid vector, before exposure to cisplatin. In vivo efficacy was determined in an intradermal RIF-1 tumour model, with intraperitoneal administration of cisplatin. Additionally, treatment potential was investigated in various human tumour cell lines including human prostate (DU145 and PC3) and human colon (HT29 and HCT116) cancer cell lines. Experimental endpoints were established using western blot, Greiss test, clonogenic assay and tumour growth delay. RESULTS: Transfection of RIF-1 tumour cells in vitro with the CMV/iNOS significantly enhanced the cytotoxicity of cisplatin (0.2-1.0 microM). In vivo transfer of CMV/iNOS by direct injection into established RIF-1 tumours caused a significant (p = 0.0027) delay in tumour growth. CMV/iNOS gene transfer in vitro resulted in the strong expression of iNOS DNA in all cell lines, and significantly increased levels of NO. in all cell lines except HCT116. CONCLUSIONS: Significant chemosensitization of cisplatin cytotoxicity was observed in the presence of NO. derived from the overexpression iNOS. We conclude that p53 status of the various cell lines was unlikely to be responsible for cisplatin-induced apoptosis.


Assuntos
Antineoplásicos/toxicidade , Cisplatino/toxicidade , Óxido Nítrico Sintase/genética , Animais , Apoptose , Linhagem Celular Tumoral , Terapia Combinada , Terapia Genética , Vetores Genéticos/genética , Humanos , Camundongos , Neoplasias Induzidas por Radiação/terapia , Transdução Genética , Transfecção
19.
Clin Cancer Res ; 14(5): 1502-9, 2008 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-18316575

RESUMO

PURPOSE: A number of cytotoxic chemotherapy agents tested at low concentrations show antiangiogenic properties with limited cytotoxicity, e.g., cyclophosphamide, tirapazamine, and mitoxantrone. AQ4N is a bioreductive alkylaminoanthraquinone that is cytotoxic when reduced to AQ4; hence, it can be used to target hypoxic tumor cells. AQ4N is structurally similar to mitoxantrone and was evaluated for antiangiogenic properties without the need for bioreduction. EXPERIMENTAL DESIGN: The effect of AQ4N and fumagillin on human microvascular endothelial cells (HMEC-1) was measured using a variety of in vitro assays, i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound scrape, tubule formation, rat aortic ring, and invasion assays. Low-dose AQ4N (20 mg/kg) was also given in vivo to mice bearing a tumor in a dorsal skin flap. RESULTS: AQ4N (10(-11) to 10(-5) mol/L) had no effect on HMEC-1 viability. AQ4N (10(-9) to 10(-5)mol/L) caused a sigmoidal dose-dependent inhibition of endothelial cell migration in the wound scrape model. Fumagillin showed a similar response over a lower dose range (10(-13) to 10(-9) mol/L); however, the maximal inhibition was less (25% versus 43% for AQ4N). AQ4N inhibited HMEC-1 cell contacts on Matrigel (10(-8) to 10(-5) mol/L), HMEC-1 cell invasion, and sprouting in rat aorta explants. Immunofluorescence staining with tubulin, vimentim, dynein, and phalloidin revealed that AQ4N caused disruption to the cell cytoskeleton. When AQ4N (20 mg/kg) was given in vivo for 5 days, microvessels disappeared in LNCaP tumors grown in a dorsal skin flap. CONCLUSIONS: This combination of assays has shown that AQ4N possesses antiangiogenic effects in normoxic conditions, which could potentially contribute to antitumor activity.


Assuntos
Inibidores da Angiogênese/farmacologia , Antraquinonas/farmacologia , Endotélio Vascular/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Pele/irrigação sanguínea , Animais , Antineoplásicos/farmacologia , Aorta/efeitos dos fármacos , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Colágeno/metabolismo , Neoplasias do Colo/tratamento farmacológico , Cicloexanos/farmacologia , Citoesqueleto/metabolismo , Combinação de Medicamentos , Ácidos Graxos Insaturados/farmacologia , Humanos , Laminina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microcirculação , Neovascularização Fisiológica , Neoplasias da Próstata/tratamento farmacológico , Proteoglicanas/metabolismo , Ratos , Ratos Wistar , Sesquiterpenos/farmacologia , Retalhos Cirúrgicos , Cicatrização/efeitos dos fármacos
20.
Int J Cancer ; 123(4): 760-8, 2008 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-18512241

RESUMO

Hypoxia confers resistance to common cancer therapies, however, it has also has been shown to result in genetic alterations which may allow a survival advantage and increase the tumorigenic properties of cancer cells. Additionally, it may exert a selection pressure, allowing expansion of tumor cells with a more aggressive phenotype. To further assess the role of hypoxia in malignant progression in prostate cancer we exposed human androgen dependent prostate cancer cells (LNCaP) to cycles of chronic hypoxia and isolated a subline, LNCaP-H1. This article describes the partial characterization of this cell line. The LNCaP-H1 subline showed altered growth characteristics and exhibited androgen independent growth both in vitro and in vivo. Furthermore, these cells were resistant to mitochondrial-mediated apoptosis, probably since the endogenous levels of Bax was lower and Bcl-2 higher than in the parental LNCaP cells. Microarray analysis revealed that a complex array of pathways had differential gene expression between the 2 cell lines, with LNCaP-H1 cells exhibiting a genetic profile which suggests that they may be more likely metastasize to distant organs, especially bone. This was supported by an in vitro invasion assay, and an in vivo metastasis study. This study shows that hypoxia can select for androgen independent prostate cancer cells which have a survival advantage and are more likely to invade and metastasize.


Assuntos
Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Androgênios/metabolismo , Animais , Apoptose/fisiologia , Processos de Crescimento Celular/fisiologia , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Expressão Gênica , Genótipo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Mitocôndrias/fisiologia , Transplante de Neoplasias , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Fenótipo , Neoplasias da Próstata/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Androgênicos/biossíntese , Receptores Androgênicos/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transplante Heterólogo
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