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1.
Theranostics ; 10(7): 3064-3082, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32194855

RESUMO

A successful matching of a PEG group size with the EPR effect for an off-to-on responsive NIR-fluorophore conjugate has been accomplished which allows two distinct in vivo tumor imaging periods, the first being the switch on during the initial tumor uptake via enhanced permeability into the ROI (as background is suppressed) and a second, later, due to enhanced retention within the tumor. Methods: Software simulation (https://mihaitodor.github.io/particle_simulation/index.html), synthetic chemistry, with in vitro and in vivo imaging have been synergistically employed to identify an optimal PEG conjugate of a bio-responsive NIR-AZA fluorophore for in vivo tumor imaging. Results: A bio-responsive NIR-AZA fluorophore conjugated to a 10 kDa PEG group has shown excellent in vivo imaging performance with sustained high tumor to background ratios and peak tumor emission within 24 h. Analysis of fluorescence profiles over 7 days has provided evidence for the EPR effect playing a positive role. Conclusion: Preclinical results show that exploiting the EPR effect by utilizing an optimized PEG substituent on a bio-responsive fluorophore may offer a means for intraoperative tumor margin delineation. The off-to-on responsive nature of the fluorophore makes tumor imaging achievable without waiting for clearance from normal tissue.

2.
Cell Death Dis ; 11(2): 124, 2020 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054850

RESUMO

Despite the introduction of novel targeted therapies, chemotherapy still remains the primary treatment for metastatic melanoma in poorly funded healthcare environments or in case of disease relapse, with no reliable molecular markers for progression-free survival (PFS) available. As chemotherapy primarily eliminates cancer cells by apoptosis, we here evaluated if the expression of key apoptosis regulators (Bax, Bak, Bcl-2, Bcl-xL, Smac, Procaspase-9, Apaf-1, Procaspase-3 and XIAP) allows prognosticating PFS in stage III/IV melanoma patients. Following antibody validation, marker expression was determined by automated and manual scoring of immunohistochemically stained tissue microarrays (TMAs) constructed from treatment-naive metastatic melanoma biopsies. Interestingly and counter-intuitively, low expression of the pro-apoptotic proteins Bax, Bak and Smac indicated better prognosis (log-rank p < 0.0001, p = 0.0301 and p = 0.0227 for automated and p = 0.0422, p = 0.0410 and p = 0.0073 for manual scoring). These findings were independently validated in the cancer genome atlas (TCGA) metastatic melanoma cohort (TCGA-SKCM) at transcript level (log-rank p = 0.0004, p = 0.0104 and p = 0.0377). Taking expression heterogeneity between the markers in individual tumour samples into account allowed defining combinatorial Bax, Bak, Smac signatures that were associated with significantly increased PFS (p = 0.0002 and p = 0.0028 at protein and transcript level, respectively). Furthermore, combined low expression of Bax, Bak and Smac allowed predicting prolonged PFS (> 12 months) on a case-by-case basis (area under the receiver operating characteristic curve (ROC AUC) = 0.79). Taken together, our results therefore suggest that Bax, Bak and Smac jointly define a signature with potential clinical utility in chemotherapy-treated metastatic melanoma.

3.
Epigenetics ; : 1-13, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32000564

RESUMO

BACKGROUND: Liquid biopsies offer significant potential for informing on cancer progression and therapeutic resistance via minimally invasive serial monitoring of genetic alterations. Although the cancer epigenome is a central driving force in most neoplasia, the accuracy of monitoring the tumor methylome using liquid biopsies remains relatively unknown. OBJECTIVES: to investigate how well two types of liquid biopsy (urine and blood) capture the prostate cancer methylome, and may thus serve as a non-invasive surrogate for studying the tumor epigenome. METHODS: A cohort of four metastatic treatment naïve prostate cancer (PCa) patients was selected. Matched biopsy cores (tumor and histologically matched-normal), post-DRE, pre-biopsy urine, and peripheral blood plasma were available for each subject. DNA methylation was profiled utilizing the Infinium® MethylationEPIC BeadChip (Illumina) and analysed using the RnBeads software. Significantly (FDR adjusted P < 0.05) differentially methylated probes (DMPs) between tumor and MN were identified and examined in the liquids (done at a grouped and individual subject level). RESULTS: DNA methylation analysis of urine and blood in men with metastatic PCa showed highly correlated patterns between the different liquid types (ρ = 0.93, P < 0.0001), with large contributions from non-tumor sources. DNA methylation profiles of liquids were more similar between subjects, than intra-individual liquid-tumor correlations. Overall, both urine and plasma are viable surrogates for tumor tissue biopsies, capturing up to 39.40% and 64.14% of tumor-specific methylation alterations, respectively. CONCLUSION: We conclude that both urine and blood plasma are easily accessible and sensitive biofluids for the study of PCa epigenomic alterations.

4.
Clin Cancer Res ; 26(3): 623-631, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31641007

RESUMO

PURPOSE: To test the validity of OncoMasTR Molecular Score (OMm), OMclin1, and OncoMasTR Risk Score (OMclin2) prognostic scores for prediction of distant recurrence (DR) in estrogen receptor (ER)-positive/HER2-negative breast cancer treated with 5 years' endocrine therapy only and compare their performance with the Oncotype DX Recurrence Score (RS). EXPERIMENTAL DESIGN: OMm incorporates three master transcription regulator genes. OMclin1 combines OMm, tumor size, grade, and nodal status; OMclin2 incorporates OMm, tumor size, and nodal status. OMclin1 and OMclin2 were evaluated for 646 postmenopausal patients with ER-positive/HER2-negative primary breast cancer with 0-3 involved lymph nodes in TransATAC. Patients were randomized to 5 years' anastrozole or tamoxifen without chemotherapy. RS was available in all cases. We used likelihood ratio-χ 2, C-index, and Kaplan-Meier analyses to assess prognostic information. RESULTS: OMm, OMclin1, and OMclin2 were highly prognostic for prediction of DR in years 0-10 among all patients [likelihood ratio (LR)-χ 2 = 25.4, 48.7, and 45.0, respectively, all P < 0.001; C-index = 0.67, 0.71, and 0.71, respectively], compared with RS (LR-χ 2 = 18.8; P < 0.001; C-index = 0.63). All three scores provided significant additional prognostic value beyond clinical treatment score, Nottingham Prognostic Index, and Ki67. OMclin1 and OMclin2 categorized 190 and 267 node-negative patients as low risk (DR rates: 2.9% and 4.9%, respectively). In comparison, RS categorized 296 node-negative patients as low-risk and 128 patients as intermediate-risk (DR rate: 6.6% and 17.3%, respectively). CONCLUSIONS: OMm, OMclin1, and OMclin2 were highly prognostic for early and late DR in women with early-stage ER-positive breast cancer receiving 5 years' endocrine therapy. In TransATAC, OMclin1 and the OncoMasTR Risk Score (OMclin2) were superior to RS in identifying patients at increased risk of DR.

5.
Chem Sci ; 10(29): 6944-6956, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31588261

RESUMO

The use of NIR-fluorescence imaging to demarcate tumour boundaries for real-time guidance of their surgical resection has a huge untapped potential. However, fluorescence imaging using molecular fluorophores, even with a targeting biomolecule attached, has a major shortcoming of signal interference from non-specific background fluorescence outside the region of interest. This poor selectivity necessitates prolonged time delays to allow clearance of background fluorophore and retention within the tumour prior to image acquisition. In this report, an innovative approach to overcome this issue is described in which cancer targeted off to on bio-responsive NIR-fluorophores are utilised to switch-on first within the tumour. Bio-responsive cRGD, iRGD and PEG conjugates have been synthesised using activated ester/amine or maleimide/thiol couplings to link targeting and fluorophore components. Their off to on emission responses were measured and compared with an always-on non-responsive control with each bio-responsive derivative showing large fluorescence enhancement values. Live cell imaging experiments using metastatic breast cancer cells confirmed in vitro bio-responsive capabilities. An in vivo assessment of MDA-MB 231 tumour imaging performance for bio-responsive and always-on fluorophores was conducted with monitoring of fluorescence distributions over 96 h. As anticipated, the always-on fluorophore gave an immediate, non-specific and very strong emission throughout whereas the bio-responsive derivatives initially displayed very low fluorescence. All three bio-responsive derivatives switched on within tumours at time points consistent with their conjugated targeting groups. cRGD and iRGD conjugates both had effective tumour turn-on in the first hour, though the cRGD derivative had superior specificity for tumour over the iRGD conjugate. The pegylated derivative had similar switch-on characteristics but over a much longer period, taking 9 h before a significant emission was observable from the tumour. Evidence for in vivo active tumour targeting was obtained for the best performing cRGD bio-responsive NIR-AZA derivative from competitive binding studies. Overall, this cRGD-conjugate has the potential to overcome the inherent drawback of targeted always-on fluorophores requiring prolonged clearance times and shows excellent potential for clinical translation for intraoperative use in fluorescence guided tumour resections.

6.
Clin Cancer Res ; 25(23): 7139-7150, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31409615

RESUMO

PURPOSE: Invasive lobular carcinoma (ILC) is a subtype of breast cancer accounting for 10% of breast tumors. The majority of patients are treated with endocrine therapy; however, endocrine resistance is common in estrogen receptor-positive breast cancer and new therapeutic strategies are needed. Bromodomain and extraterminal inhibitors (BETi) are effective in diverse types of breast cancer but they have not yet been assessed in ILC. EXPERIMENTAL DESIGN: We assessed whether targeting the BET proteins with JQ1 could serve as an effective therapeutic strategy in ILC in both 2D and 3D models. We used dynamic BH3 profiling and RNA-sequencing (RNA-seq) to identify transcriptional reprograming enabling resistance to JQ1-induced apoptosis. As part of the RATHER study, we obtained copy-number alterations and RNA-seq on 61 ILC patient samples. RESULTS: Certain ILC cell lines were sensitive to JQ1, while others were intrinsically resistant to JQ1-induced apoptosis. JQ1 treatment led to an enhanced dependence on antiapoptotic proteins and a transcriptional rewiring inducing fibroblast growth factor receptor 1 (FGFR1). This increase in FGFR1 was also evident in invasive ductal carcinoma (IDC) cell lines. The combination of JQ1 and FGFR1 inhibitors was highly effective at inhibiting growth in both 2D and 3D models of ILC and IDC. Interestingly, we found in the RATHER cohort of 61 ILC patients that 20% had FGFR1 amplification and we showed that high BRD3 mRNA expression was associated with poor survival specifically in ILC. CONCLUSIONS: We provide evidence that BETi either alone or in combination with FGFR1 inhibitors or BH3 mimetics may be a useful therapeutic strategy for recurrent ILC patients.

7.
Oncotarget ; 10(38): 3725-3744, 2019 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-31217905

RESUMO

Colorectal cancer (CRC) is the second leading cause of cancer associated deaths in developed countries. Cancer progression and metastatic spread is reliant on new blood vasculature, or angiogenesis. Tumour-related angiogenesis is regulated by pro- and anti-angiogenic factors secreted from malignant tissue in a stepwise process. Previously we structurally modified the small anti-angiogenic molecule quininib and discovered a more potent anti-angiogenic compound 1, 4 dihydroxy quininib (Q8), an antagonist of cysteinyl leukotriene receptor-1 with VEGF-independent bioactivity. Here, Q8, quininib (Q1) and five structural analogues were assayed for anti-tumorigenic effects in pre-clinical cancer models. Q8 reduced clone formation of the human colorectal cancer cell line HT29-Luc2. Gene silencing of CysLT1 in HT29-Luc2 cells significantly reduced expression of calpain-2. In human ex vivo colorectal cancer tumour explants, Q8 significantly decreased the secretion of both TIE-2 and VCAM-1 expression. In vivo Q8 was well tolerated up to 50 mg/kg by Balb/C mice and significantly more effective at reducing tumour volume in colorectal tumour xenografts compared to the parent drug quininib. In tumour xenografts, Q8 significantly reduced expression of the angiogenic marker calpain-2. In summary, we propose Q8 may act on the TIE-2-Angiopoietin signalling pathway to significantly inhibit the process of tumour angiogenesis in colorectal cancer.

8.
Sci Rep ; 9(1): 9204, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235775

RESUMO

Angiogenesis is a key tumor microenvironment (TME) event underpinning tumor growth and metastasis. Nevertheless, the relatively poor performance of anti-angiogenic therapies in clinical trials compared to pre-clinical studies implies that classical subcutaneous xenograft models have limited predictive potential in this setting. To address this issue, we established orthotopic surgical resection models of breast cancer, which replicate the phenotype of clinical post-resection micro-metastasis. To demonstrate the power and precision of these models, we recapitulated the BETH adjuvant trial (NCT00625898) where the addition of bevacizumab (BVZ) to chemotherapy plus trastuzumab (Trast) failed to provide additional benefit. SCID mice were orthotopically implanted with bioluminescent Her2+ MDA-MB-231 or HCC1954 cells and tumors resected c.5 weeks later. Following resection, mice were treated with 10 mg/kg Trast +5 mg/kg paclitaxel (PAC) IP once weekly for 6 cycles +/- weekly BVZ (5 mg/kg IP). Metastasis was monitored by imaging. Using these models our data confirms that the addition of the anti-angiogenic antibody BVZ to adjuvant Trast + chemotherapy provides no additional benefit compared with Trast + chemotherapy alone. Previous studies using non-resection subcutaneously engrafted xenografts failed to predict this outcome. Our results provide compelling evidence for the utility of cell line xenograft resection models to predict clinical outcome for TME targeting agents.

10.
JCO Clin Cancer Inform ; 3: 1-17, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30995124

RESUMO

PURPOSE: Dynamic network models predict clinical prognosis and inform therapeutic intervention by elucidating disease-driven aberrations at the systems level. However, the personalization of model predictions requires the profiling of multiple model inputs, which hampers clinical translation. PATIENTS AND METHODS: We applied APOPTO-CELL, a prognostic model of apoptosis signaling, to showcase the establishment of computational platforms that require a reduced set of inputs. We designed two distinct and complementary pipelines: a probabilistic approach to exploit a consistent subpanel of inputs across the whole cohort (Ensemble) and a machine learning approach to identify a reduced protein set tailored for individual patients (Tree). Development was performed on a virtual cohort of 3,200,000 patients, with inputs estimated from clinically relevant protein profiles. Validation was carried out in an in-house stage III colorectal cancer cohort, with inputs profiled in surgical resections by reverse phase protein array (n = 120) and/or immunohistochemistry (n = 117). RESULTS: Ensemble and Tree reproduced APOPTO-CELL predictions in the virtual patient cohort with 92% and 99% accuracy while decreasing the number of inputs to a consistent subset of three proteins (40% reduction) or a personalized subset of 2.7 proteins on average (46% reduction), respectively. Ensemble and Tree retained prognostic utility in the in-house colorectal cancer cohort. The association between the Ensemble accuracy and prognostic value (Spearman ρ = 0.43; P = .02) provided a rationale to optimize the input composition for specific clinical settings. Comparison between profiling by reverse phase protein array (gold standard) and immunohistochemistry (clinical routine) revealed that the latter is a suitable technology to quantify model inputs. CONCLUSION: This study provides a generalizable framework to optimize the development of network-based prognostic assays and, ultimately, to facilitate their integration in the routine clinical workflow.

11.
Cancer Res ; 79(10): 2619-2633, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30885980

RESUMO

The PDLIM2 protein regulates stability of transcription factors including NF-κB and STATs in epithelial and hemopoietic cells. PDLIM2 is strongly expressed in certain cancer cell lines that exhibit an epithelial-to-mesenchymal phenotype, and its suppression is sufficient to reverse this phenotype. PDLIM2 supports the epithelial polarity of nontransformed breast cells, suggesting distinct roles in tumor suppression and oncogenesis. To better understand its overall function, we investigated PDLIM2 expression and activity in breast cancer. PDLIM2 protein was present in 60% of tumors diagnosed as triple-negative breast cancer (TNBC), and only 20% of other breast cancer subtypes. High PDLIM2 expression in TNBC was positively correlated with adhesion signaling and ß-catenin activity. Interestingly, PDLIM2 was restricted to the cytoplasm/membrane of TNBC cells and excluded from the nucleus. In breast cell lines, PDLIM2 retention in the cytoplasm was controlled by cell adhesion, and translocation to the nucleus was stimulated by insulin-like growth factor-1 or TGFß. Cytoplasmic PDLIM2 was associated with active ß-catenin and ectopic expression of PDLIM2 was sufficient to increase ß-catenin levels and its transcriptional activity in reporter assays. Suppression of PDLIM2 inhibited tumor growth in vivo, whereas overexpression of PDLIM2 disrupted growth in 3D cultures. These results suggest that PDLIM2 may serve as a predictive biomarker for a large subset of TNBC whose phenotype depends on adhesion-regulated ß-catenin activity and which may be amenable to therapies that target these pathways. SIGNIFICANCE: This study shows that PDLIM2 expression defines a subset of triple-negative breast cancer that may benefit from targeting the ß-catenin and adhesion signaling pathways. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/10/2619/F1.large.jpg.


Assuntos
Biomarcadores Tumorais/metabolismo , Adesão Celular , Proteínas com Domínio LIM/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , beta Catenina/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Feminino , Células HEK293 , Humanos
12.
Proteomics Clin Appl ; 13(4): e1800159, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30768761

RESUMO

PURPOSE: The tyrosine kinase inhibitor (TKI) sunitinib is a multi-targeted agent approved across multiple cancer indications. Nevertheless, since approval, data has emerged to describe a worrisome side effect profile including hypertension, hand-foot syndrome, fatigue, diarrhea, mucositis, proteinuria, and (rarely) congestive heart failure. It has been hypothesized that the observed multi-parameter toxicity profile is related to "on-target" kinase inhibition in "off-target" tissues. EXPERIMENTAL DESIGN: To interrogate off-target effects in pre-clinical studies, a reverse phase protein array (RPPA) approach is employed. Mice are treated with sunitinib (40 mg kg-1 ) for 4 weeks, following which critical organs are removed. The Zeptosens RPPA platform is employed for protein expression analysis. RESULTS: Differentially expressed proteins associated with damage and/or stress are found in the majority of organs from treated animals. Proteins differentially expressed in the heart are associated with myocardial hypertrophy, ischaemia/reperfusion, and hypoxia. However, hypertrophy is not evidenced on histology. Mild proteinuria is observed; however, no changes in renal glomerular structure are visible via electron microscopy. In skin, proteins associated with cutaneous inflammation, keratinocyte hyper-proliferation, and increased inflammatory response are differentially expressed. CONCLUSIONS AND CLINICAL RELEVANCE: It is posited that pre-clinical implementation of a combined histopathological/RPPA approach provides a sensitive method to mechanistically elucidate the early manifestation of TKI on-target/organ off-target toxicities.


Assuntos
Análise Serial de Proteínas , Inibidores de Proteínas Quinases/efeitos adversos , Proteoma/biossíntese , Sunitinibe/efeitos adversos , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Inibidores de Proteínas Quinases/farmacologia , Sunitinibe/farmacologia
13.
Circ Heart Fail ; 12(3): e005765, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30798618

RESUMO

BACKGROUND: Limited knowledge exists of the extent of epigenetic alterations, such as DNA methylation, in heart failure (HF). We conducted targeted DNA methylation sequencing to identify DNA methylation alterations in coding and noncoding RNA (ncRNA) across different etiological subtypes of HF. METHODS AND RESULTS: A targeted bisulfite sequence capture sequencing platform was applied to DNA extracted from cardiac interventricular septal tissue of 30 male HF patients encompassing causes including hypertrophic obstructive cardiomyopathy, ischemic cardiomyopathy, dilated cardiomyopathy, and 9 control patients with nonfailing hearts. We detected 62 678 differentially methylated regions in the studied HF cohort. By comparing each HF subgroup to the nonfailing control group, we identified 195 unique differentially methylated regions: 5 in hypertrophic obstructive cardiomyopathy, 151 in dilated cardiomyopathy, and 55 in ischemic cardiomyopathy. These translated to 4 genes/1 ncRNA in hypertrophic obstructive cardiomyopathy, 131 genes/17 ncRNA in dilated cardiomyopathy, and 51 genes/5 ncRNA in ischemic cardiomyopathy. Subsequent gene/ncRNA expression analysis was assessed using quantitative reverse transcription polymerase chain reaction and revealed 6 genes: 4 hypermethylated ( HEY2, MSR1, MYOM3, and COX17), 2 hypomethylated ( CTGF and MMP2); and 2 microRNA: 1 hypermethylated (miR-24-1), 1 hypomethylated (miR-155) with significantly upregulated or downregulated expression levels consistent with the direction of methylation in the particular HF subgroup. CONCLUSIONS: For the first time DNA methylation alterations and associated gene expression changes were identified in etiologically variant pathological HF tissue. The methylation-sensitive and disease-associated genes/ncRNA identified from this study represent a unique cohort of loci that demonstrate a plausible potential as a novel diagnostic and therapeutic target in HF and warrant further investigation.


Assuntos
Metilação de DNA/genética , Epigênese Genética/genética , Insuficiência Cardíaca/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Coração/fisiopatologia , Humanos , Masculino , Fenótipo
14.
JCO Precis Oncol ; 20192019.
Artigo em Inglês | MEDLINE | ID: mdl-30801051

RESUMO

Purpose: Liquid biopsies that noninvasively detect molecular correlates of aggressive prostate cancer (PCa) could be used to triage patients, reducing the burdens of unnecessary invasive prostate biopsy and enabling early detection of high-risk disease. DNA hypermethylation is among the earliest and most frequent aberrations in PCa. We investigated the accuracy of a six-gene DNA methylation panel (Epigenetic Cancer of the Prostate Test in Urine [epiCaPture]) at detecting PCa, high-grade (Gleason score greater than or equal to 8) and high-risk (D'Amico and Cancer of the Prostate Risk Assessment] PCa from urine. Patients and Methods: Prognostic utility of epiCaPture genes was first validated in two independent prostate tissue cohorts. epiCaPture was assessed in a multicenter prospective study of 463 men undergoing prostate biopsy. epiCaPture was performed by quantitative methylation-specific polymerase chain reaction in DNA isolated from prebiopsy urine sediments and evaluated by receiver operating characteristic and decision curves (clinical benefit). The epiCaPture score was developed and validated on a two thirds training set to one third test set. Results: Higher methylation of epiCaPture genes was significantly associated with increasing aggressiveness in PCa tissues. In urine, area under the receiver operating characteristic curve was 0.64, 0.86, and 0.83 for detecting PCa, high-grade PCa, and high-risk PCa, respectively. Decision curves revealed a net benefit across relevant threshold probabilities. Independent analysis of two epiCaPture genes in the same clinical cohort provided analytical validation. Parallel epiCaPture analysis in urine and matched biopsy cores showed added value of a liquid biopsy. Conclusion: epiCaPture is a urine DNA methylation test for high-risk PCa. Its tumor specificity out-performs that of prostate-specific antigen (greater than 3 ng/mL). Used as an adjunct to prostate-specific antigen, epiCaPture could aid patient stratification to determine need for biopsy.

15.
Eur J Med Chem ; 161: 343-353, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30368132

RESUMO

Clinical imaging utilising near-infrared fluorescence is growing as an intraoperative aid for the decision-making processes during complex surgical procedures. Existing uses include perfusion assessment and lymph node identification with many new applications currently being proposed and developed. While imaging hardware and software have significantly progressed in recent times, suitable NIR-fluorophores remain a limiting factor. In this report, we describe the design, synthesis, photophysical characterization and in vivo imaging assessment of new PEGylated NIR-fluorophores based on the BF2-azadipyrromethene fluorophore class. The synthetic route includes PEGylation as the final step, thereby allowing routine access to derivatives substituted with different molecular weights of PEG. Absorption and emission wavelength maxima in PBS lie at 690 and 720 nm respectively with quantum yields over 12%. They show excellent photostability and no light induced singlet oxygen production. A time-course of NIR-fluorescence imaging, post i.v. administration, in BALB/c mice showed a rapid and preferential accumulation in the renal excretion pathway within 20 min, indicative of potential clinical usage for intraoperative identification of vial structures along this pathway. Assessment with clinical imaging equipment showed the NIR-AZA fluorophores to be wavelength compatible and brighter than currently used methylene blue (MB), and that they have the ability to be imaged simultaneously with indocyanine green (ICG) offering a potential for dual colour clinical imaging.


Assuntos
Compostos Aza/química , Compostos de Boro/química , Neoplasias da Mama/diagnóstico por imagem , Corantes Fluorescentes/química , Imagem Óptica , Polietilenoglicóis/química , Porfobilinogênio/análogos & derivados , Animais , Compostos Aza/administração & dosagem , Compostos de Boro/administração & dosagem , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/síntese química , Humanos , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Polietilenoglicóis/administração & dosagem , Porfobilinogênio/administração & dosagem , Porfobilinogênio/química , Eliminação Renal , Espectrometria de Fluorescência , Relação Estrutura-Atividade
16.
BMC Cancer ; 18(1): 965, 2018 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-30305055

RESUMO

BACKGROUND: Lapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells. METHODS: We examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer. RESULTS: In this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells. CONCLUSIONS: Our findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Lapatinib/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Proteína Forkhead Box O3/biossíntese , Expressão Gênica/efeitos dos fármacos , Genes erbB-2 , Humanos , Lapatinib/uso terapêutico , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico
17.
Nat Commun ; 9(1): 4112, 2018 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-30291241

RESUMO

Increased copy number alterations (CNAs) indicative of chromosomal instability (CIN) have been associated with poor cancer outcome. Here, we study CNAs as potential biomarkers of bevacizumab (BVZ) response in metastatic colorectal cancer (mCRC). We cluster 409 mCRCs in three subclusters characterized by different degrees of CIN. Tumors belonging to intermediate-to-high instability clusters have improved outcome following chemotherapy plus BVZ versus chemotherapy alone. In contrast, low instability tumors, which amongst others consist of POLE-mutated and microsatellite-instable tumors, derive no further benefit from BVZ. This is confirmed in 81 mCRC tumors from the phase 2 MoMa study involving BVZ. CNA clusters overlap with CRC consensus molecular subtypes (CMS); CMS2/4 xenografts correspond to intermediate-to-high instability clusters and respond to FOLFOX chemotherapy plus mouse avastin (B20), while CMS1/3 xenografts match with low instability clusters and fail to respond. Overall, we identify copy number load as a novel potential predictive biomarker of BVZ combination therapy.


Assuntos
Adenocarcinoma/genética , Antineoplásicos Imunológicos/uso terapêutico , Bevacizumab/uso terapêutico , Neoplasias Colorretais/genética , Variações do Número de Cópias de DNA , Adenocarcinoma/tratamento farmacológico , Idoso , Animais , Instabilidade Cromossômica , Neoplasias Colorretais/tratamento farmacológico , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Estudos Retrospectivos , Ensaios Antitumorais Modelo de Xenoenxerto
18.
J Mol Med (Berl) ; 96(10): 1025-1037, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30069746

RESUMO

Elevated levels of the anti-apoptotic BCL2 protein associate with favourable outcome in breast cancer. We investigated whether executioner caspase activation downstream of mitochondrial apoptosis was associated with, or independent, of BCL2's prognostic signature in breast cancer. Levels of pro- and anti-apoptotic BCL2 family proteins were quantified in triple negative breast cancer (TNBC) samples and utilised to calculate BCL2 profiles of 845 breast cancer patients. Biomarkers including single apoptosis proteins and network-enriched apoptosis system signatures were evaluated using uni- and multi-variate Cox-models. In both TNBC and non-TNBC breast cancer, the anti-apoptotic BCL2 protein was particularly abundant when compared to other solid tumours. High BCL2 protein levels were prognostic of favourable outcome across all breast cancers (HR 0.4, 95% CI 0.2-0.6, Wald p < 0.0001). Although BCL2 and cleaved caspase-7 levels were negatively correlated, levels of cleaved caspase-7 were also associated with favourable outcome (HR 0.4, 95% CI 0.3-0.7, Wald p = 0.001). A combination of low BCL2 and low cleaved caspase-7 protein levels was highly prognostic of unfavourable outcome across all breast cancers (HR 11.29, 95% CI 2.20-58.23, Wald p = 0.01). A combination of BCL2 and cleaved caspase-7 levels is a promising prognostic biomarker in breast cancer patients. KEY MESSAGE: BCL2 levels are elevated in breast cancer where they are marker of good prognosis. BCL2 and active caspase levels correlate negatively; yet, active caspases indicate good outcome. Low BCL2 and low caspase-7 are highly prognostic of unfavourable outcome across all breast cancers. BCL2 levels indicate molecular subtype and tumour proliferation status in breast cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Caspase 7/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Feminino , Humanos , Prognóstico
19.
J Clin Oncol ; 36(20): 2052-2060, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-29792754

RESUMO

Purpose Patients with metastatic colorectal cancer (mCRC) have limited benefit from the addition of bevacizumab to standard chemotherapy. However, a subset probably benefits substantially, highlighting an unmet clinical need for a biomarker of response to bevacizumab. Previously, we demonstrated that losses of chromosomes 5q34, 17q12, and 18q11.2-q12.1 had a significant correlation with progression-free survival (PFS) in patients with mCRC treated with bevacizumab in the CAIRO2 clinical trial but not in patients who did not receive bevacizumab in the CAIRO trial. This study was designed to validate these findings. Materials and Methods Primary mCRC samples were analyzed from two cohorts of patients who received bevacizumab as first-line treatment; 96 samples from the European multicenter study Angiopredict (APD) and 81 samples from the Italian multicenter study, MOMA. A third cohort of 90 samples from patients with mCRC who did not receive bevacizumab was analyzed. Copy number aberrations of tumor biopsy specimens were measured by shallow whole-genome sequencing and were correlated with PFS, overall survival (OS), and response. Results Loss of chromosome 18q11.2-q12.1 was associated with prolonged PFS most significantly in both the cohorts that received bevacizumab (APD: hazard ratio, 0.54; P = .01; PFS difference, 65 days; MOMA: hazard ratio, 0.55; P = .019; PFS difference, 49 days). A similar association was found for OS and overall response rate in these two cohorts, which became significant when combined with the CAIRO2 cohort. Median PFS in the cohort of patients with mCRC who did not receive bevacizumab and in the CAIRO cohort was similar to that of the APD, MOMA, and CAIRO2 patients without an 18q11.2-q12.1 loss. Conclusion We conclude that the loss of chromosome 18q11.2-q12.1 is consistently predictive for prolonged PFS in patients receiving bevacizumab. The predictive value of this loss is substantiated by a significant gain in OS and overall response rate.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Deleção Cromossômica , Cromossomos Humanos Par 18 , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Bevacizumab/administração & dosagem , Capecitabina/administração & dosagem , Cromossomos Humanos Par 17 , Cromossomos Humanos Par 5 , Ensaios Clínicos Fase III como Assunto , Estudos de Coortes , Neoplasias Colorretais/patologia , Hibridização Genômica Comparativa , Feminino , Testes Genéticos , Humanos , Irinotecano/administração & dosagem , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Oxaliplatina/administração & dosagem , Intervalo Livre de Progressão , Reprodutibilidade dos Testes
20.
Oncotarget ; 9(21): 15673-15690, 2018 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-29644001

RESUMO

Triple-negative breast cancer (TNBC), is a heterogeneous disease characterised by absence of expression of the estrogen receptor (ER), progesterone receptor (PR) and lack of amplification of human epidermal growth factor receptor 2 (HER2). TNBC patients can exhibit poor prognosis and high recurrence stages despite early response to chemotherapy treatment. In this study, we identified a pro-survival signalling protein BCL2- associated athanogene 3 (BAG3) to be highly expressed in a subset of TNBC cell lines and tumour tissues. High mRNA expression of BAG3 in TNBC patient cohorts significantly associated with a lower recurrence free survival. The epidermal growth factor receptor (EGFR) is amplified in TNBC and EGFR signalling dynamics impinge on cancer cell survival and disease recurrence. We found a correlation between BAG3 and EGFR expression in TNBC cell lines and determined that BAG3 can regulate tumour cell proliferation, migration and invasion in EGFR expressing TNBC cells lines. We identified an interaction between BAG3 and components of the EGFR signalling networks using mass spectrometry. Furthermore, BAG3 contributed to regulation of proliferation in TNBC cell lines by reducing the activation of components of the PI3K/AKT and FAK/Src signalling subnetworks. Finally, we found that combined targeting of BAG3 and EGFR was more effective than inhibition of EGFR with Cetuximab alone in TNBC cell lines. This study demonstrates a role for BAG3 in regulation of distinct EGFR modules and highlights the potential of BAG3 as a therapeutic target in TNBC.

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