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1.
Mol Oncol ; 14(1): 22-41, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31733171

RESUMO

Ultraviolet radiation-induced DNA mutations are a primary environmental driver of melanoma. The reason for this very high level of unrepaired DNA lesions leading to these mutations is still poorly understood. The primary DNA repair mechanism for UV-induced lesions, that is, the nucleotide excision repair pathway, appears intact in most melanomas. We have previously reported a postreplication repair mechanism that is commonly defective in melanoma cell lines. Here we have used a genome-wide approach to identify the components of this postreplication repair mechanism. We have used differential transcript polysome loading to identify transcripts that are associated with UV response, and then functionally assessed these to identify novel components of this repair and cell cycle checkpoint network. We have identified multiple interaction nodes, including global genomic nucleotide excision repair and homologous recombination repair, and previously unexpected MASTL pathway, as components of the response. Finally, we have used bioinformatics to assess the contribution of dysregulated expression of these pathways to the UV signature mutation load of a large melanoma cohort. We show that dysregulation of the pathway, especially the DNA damage repair components, are significant contributors to UV mutation load, and that dysregulation of the MASTL pathway appears to be a significant contributor to high UV signature mutation load.

3.
J Cell Sci ; 132(24)2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31767623

RESUMO

Melanocytic cell interactions are integral to skin homeostasis, and affect the outcome of multiple diseases, including cutaneous pigmentation disorders and melanoma. By using automated-microscopy and machine-learning-assisted morphology analysis of primary human melanocytes in co-culture, we performed combinatorial interrogation of melanocyte genotypic variants and functional assessment of lentivirus-introduced mutations. Keratinocyte-induced melanocyte dendricity, an indicator of melanocyte differentiation, was reduced in the melanocortin 1 receptor (MC1R) R/R variant strain and by NRAS.Q61K and BRAF.V600E expression, while expression of CDK4.R24C and RAC1.P29S had no detectable effect. Time-lapse tracking of melanocytes in co-culture revealed dynamic interaction phenotypes and hyper-motile cell states that indicated that, in addition to the known role in activating mitogenic signalling, MEK-pathway-activating mutations may also allow melanocytes to escape keratinocyte control and increase their invasive potential. Expanding this combinatorial platform will identify other therapeutic target mutations and melanocyte genetic variants, as well as increase understanding of skin cell interactions.

4.
Cancers (Basel) ; 11(9)2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31500184

RESUMO

The poor selectivity of standard cytotoxic chemotherapy regimens causes severe side-effects in patients and reduces the quality of life during treatment. Targeting cancer-specific vulnerabilities can improve response rates, increase overall survival and limit toxic side effects in patients. Oncogene-induced replication stress serves as a tumour specific vulnerability and rationale for the clinical development of inhibitors targeting the DNA damage response (DDR) kinases (CHK1, ATR, ATM and WEE1). CHK1 inhibitors (CHK1i) have served as the pilot compounds in this class and their efficacy in clinical trials as single agents has been disappointing. Initial attempts to combine CHK1i with chemotherapies agents that enhance replication stress (such as gemcitabine) were reported to be excessively toxic. More recently, it has emerged that combining CHK1i with subclinical doses of replication stress inducers is more effective, better tolerated and more compatible with immunotherapies. Here we focus on the lessons learned during the clinical development of CHK1i with the goal of improving the design of future clinical trials utilizing DDR inhibitors to target replication stress in cancer.

5.
Mol Oncol ; 13(7): 1503-1518, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31044505

RESUMO

Drugs such as gemcitabine that increase replication stress are effective chemotherapeutics in a range of cancer settings. These drugs effectively block replication and promote DNA damage, triggering a cell cycle checkpoint response through the ATR-CHK1 pathway. Inhibiting this signalling pathway sensitises cells to killing by replication stress-inducing drugs. Here, we investigated the effect of low-level replication stress induced by low concentrations (> 0.2 mm) of the reversible ribonucleotide reductase inhibitor hydroxyurea (HU), which slows S-phase progression but has little effect on cell viability or proliferation. We demonstrate that HU effectively synergises with CHK1, but not ATR inhibition, in > 70% of melanoma and non-small-cell lung cancer cells assessed, resulting in apoptosis and complete loss of proliferative potential in vitro and in vivo. Normal fibroblasts and haemopoietic cells retain viability and proliferative potential following exposure to CHK1 inhibitor plus low doses of HU, but normal cells exposed to CHK1 inhibitor combined with submicromolar concentrations of gemcitabine exhibited complete loss of proliferative potential. The effects of gemcitabine on normal tissue correlate with irreversible ATR-CHK1 pathway activation, whereas low doses of HU reversibly activate CHK1 independently of ATR. The combined use of CHK1 inhibitor and subclinical HU also triggered an inflammatory response involving the recruitment of macrophages in vivo. These data indicate that combining CHK1 inhibitor with subclinical HU is superior to combination with gemcitabine, as it provides equal anticancer efficacy but with reduced normal tissue toxicity. These data suggest a significant proportion of melanoma and lung cancer patients could benefit from treatment with this drug combination.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Hidroxiureia/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Animais , Antimetabólitos Antineoplásicos/efeitos adversos , Antimetabólitos Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Desoxicitidina/efeitos adversos , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Progressão da Doença , Feminino , Humanos , Hidroxiureia/efeitos adversos , Hidroxiureia/uso terapêutico , Neoplasias Pulmonares/patologia , Melanoma/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/uso terapêutico
7.
Oral Oncol ; 86: 105-112, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30409290

RESUMO

OBJECTIVES: Human papilloma virus (HPV) is the main culprit in cancers of the cervix, penis, anus, skin, eye and head and neck. Current treatments for HPV cancers have not altered survival outcomes for 30 years and there is a significant lack of targeted therapeutic agents in the management of advanced HPV-related HNSCC. Here we show that survival and maintenance of HPV-positive HNC cells relies on the continuous expression of the major HPV oncogene, E7, and that Aurora kinases are critical for survival of high-risk HPV-positive HNC cells. MATERIALS AND METHODS: To assess the role of HPV E7 on HNC cell survival, RNA interference (RNAi) of the E7 gene was initially performed. Using an Aurora kinase inhibitor, Alisertib, the role of Aurora kinases in the carcinogenesis of HPV E7 positive HNC tumour lines was then investigated. An in vivo HNC xenograft model was also utilised to assess loss of tumour volume in response to RNAi E7 gene silencing and Alisertib treatment. RESULTS: RNAi silencing of the HPV E7 gene inhibited the growth of HPV-positive HNC cells and in vivo tumour load. We show that HPV E7 oncogene expression confers sensitivity to Alisertib on HNC cells where Alisertib-mediated loss in in vitro cell viability and in vivo tumour load is dependent on E7 expression. Moreover, Aurora kinase inhibition induced degradation of MCL-1 in HPV E7-expressing HNC cells. CONCLUSION: Overall, we show that Aurora kinases are a novel therapeutic target for HPV-positive HNCs. It might be feasible to combine Aurora kinase and MCL-1 inhibitors for future HNC therapies.


Assuntos
Aurora Quinase A/antagonistas & inibidores , Aurora Quinase B/antagonistas & inibidores , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Aurora Quinase A/metabolismo , Aurora Quinase B/metabolismo , Azepinas/farmacologia , Azepinas/uso terapêutico , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/virologia , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 16/metabolismo , Humanos , Leupeptinas/farmacologia , Leupeptinas/uso terapêutico , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Proteólise/efeitos dos fármacos , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Interferência de RNA , Ensaios Antitumorais Modelo de Xenoenxerto
8.
EMBO Mol Med ; 10(9)2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30108112

RESUMO

The centrosomal protein, CEP55, is a key regulator of cytokinesis, and its overexpression is linked to genomic instability, a hallmark of cancer. However, the mechanism by which it mediates genomic instability remains elusive. Here, we showed that CEP55 overexpression/knockdown impacts survival of aneuploid cells. Loss of CEP55 sensitizes breast cancer cells to anti-mitotic agents through premature CDK1/cyclin B activation and CDK1 caspase-dependent mitotic cell death. Further, we showed that CEP55 is a downstream effector of the MEK1/2-MYC axis. Blocking MEK1/2-PLK1 signaling therefore reduced outgrowth of basal-like syngeneic and human breast tumors in in vivo models. In conclusion, high CEP55 levels dictate cell fate during perturbed mitosis. Forced mitotic cell death by blocking MEK1/2-PLK1 represents a potential therapeutic strategy for MYC-CEP55-dependent basal-like, triple-negative breast cancers.


Assuntos
Aneuploidia , Proteínas de Ciclo Celular/metabolismo , Citocinese , Mitose , Proteínas Nucleares/metabolismo , Neoplasias da Mama/patologia , Proteína Quinase CDC2/metabolismo , Caspases/metabolismo , Proteínas de Ciclo Celular/genética , Morte Celular , Linhagem Celular Tumoral , Ciclina B/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Modelos Biológicos , Proteínas Nucleares/genética
9.
Oncotarget ; 9(9): 8206-8222, 2018 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-29492189

RESUMO

Besides somatic mutations or drug efflux, epigenetic reprogramming can lead to acquired drug resistance. We recently have identified early stress-induced multi-drug tolerant cancer cells termed induced drug-tolerant cells (IDTCs). Here, IDTCs were generated using different types of cancer cell lines; melanoma, lung, breast and colon cancer. A common loss of the H3K4me3 and H3K27me3 and gain of H3K9me3 mark was observed as a significant response to drug exposure or nutrient starvation in IDTCs. These epigenetic changes were reversible upon drug holidays. Microarray, qRT-PCR and protein expression data confirmed the up-regulation of histone methyltransferases (SETDB1 and SETDB2) which contribute to the accumulation of H3K9me3 concomitantly in the different cancer types. Genome-wide studies suggest that transcriptional repression of genes is due to concordant loss of H3K4me3 and regional increment of H3K9me3. Conversely, genome-wide CpG site-specific DNA methylation showed no common changes at the IDTC state. This suggests that distinct histone methylation patterns rather than DNA methylation are driving the transition from parental to IDTCs. In addition, silencing of SETDB1/2 reversed multi drug tolerance. Alterations of histone marks in early multi-drug tolerance with an increment in H3K9me3 and loss of H3K4me3/H3K27me3 is neither exclusive for any particular stress response nor cancer type specific but rather a generic response.

10.
Clin Cancer Res ; 24(12): 2901-2912, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29535131

RESUMO

Purpose: Checkpoint kinase 1 inhibitors (CHEK1i) have single-agent activity in vitro and in vivo Here, we have investigated the molecular basis of this activity.Experimental Design: We have assessed a panel of melanoma cell lines for their sensitivity to the CHEK1i GNE-323 and GDC-0575 in vitro and in vivo The effects of these compounds on responses to DNA replication stress were analyzed in the hypersensitive cell lines.Results: A subset of melanoma cell lines is hypersensitive to CHEK1i-induced cell death in vitro, and the drug effectively inhibits tumor growth in vivo In the hypersensitive cell lines, GNE-323 triggers cell death without cells entering mitosis. CHEK1i treatment triggers strong RPA2 hyperphosphorylation and increased DNA damage in only hypersensitive cells. The increased replication stress was associated with a defective S-phase cell-cycle checkpoint. The number and intensity of pRPA2 Ser4/8 foci in untreated tumors appeared to be a marker of elevated replication stress correlated with sensitivity to CHEK1i.Conclusions: CHEK1i have single-agent activity in a subset of melanomas with elevated endogenous replication stress. CHEK1i treatment strongly increased this replication stress and DNA damage, and this correlated with increased cell death. The level of endogenous replication is marked by the pRPA2Ser4/8 foci in the untreated tumors, and may be a useful marker of replication stress in vivoClin Cancer Res; 24(12); 2901-12. ©2018 AACR.


Assuntos
Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Replicação do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Melanoma/genética , Melanoma/metabolismo , Estresse Fisiológico , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos , Mitose/efeitos dos fármacos , Mitose/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Sci Rep ; 8(1): 5144, 2018 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-29572477

RESUMO

Successive rounds of chemical modification in three generations of benzopyran molecules have shown to select for different mechanisms of actions and progressive increases in anti-cancer activity. In this study, we investigated the mechanism of action of the third-generation benzopyran compounds, TRX-E-002-1 and TRX-E-009-1. High-content screening of a panel of 240 cancer cell lines treated with TRX-E-009-1 demonstrated it has broad anti-cancer potential. Within this screen, melanoma cell lines showed a range of sensitivities and subsequently a second independent panel of 21 melanoma 3D spheroid lines were assessed for their responses to both TRX-E-002-1 and TRX-E-009-1 compounds. Time-lapse microscopy illustrated both of these compounds caused mitotic delays in treated cells, resulting in either mitotic slippage or apoptosis. This finding along with immunostaining, in vitro polymerization assays, and animal experiments in both athymic and immunocompetent mice, demonstrates that these third-generation benzopyran compounds are potent tubulin polymerization inhibitors in vitro and in vivo, and this is the molecular basis of their anti-cancer activity in melanoma. These findings indicate these BP compounds may offer a novel anti-microtubule strategy for cancer intervention and provides the basis for further investigation into biomarkers of clinical sensitivity.


Assuntos
Benzopiranos , Flavonoides , Melanoma Experimental/tratamento farmacológico , Moduladores de Tubulina , Animais , Benzopiranos/química , Benzopiranos/farmacologia , Linhagem Celular Tumoral , Flavonoides/química , Flavonoides/farmacologia , Humanos , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Nus , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Clin Cancer Res ; 24(5): 1090-1102, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29196297

RESUMO

Purpose: Identify and characterize novel combinations of sorafenib with anti-inflammatory painkillers to target difficult-to-treat RAS-mutant cancer.Experimental Design: The cytotoxicity of acetylsalicylic acid (aspirin) in combination with the multikinase inhibitor sorafenib (Nexavar) was assessed in RAS-mutant cell lines in vitro The underlying mechanism for the increased cytotoxicity was investigated using selective inhibitors and shRNA-mediated gene knockdown. In vitro results were confirmed in RAS-mutant xenograft mouse models in vivoResults: The addition of aspirin but not isobutylphenylpropanoic acid (ibruprofen) or celecoxib (Celebrex) significantly increased the in vitro cytotoxicity of sorafenib. Mechanistically, combined exposure resulted in increased BRAF/CRAF dimerization and the simultaneous hyperactivation of the AMPK and ERK pathways. Combining sorafenib with other AMPK activators, such as metformin or A769662, was not sufficient to decrease cell viability due to sole activation of the AMPK pathway. The cytotoxicity of sorafenib and aspirin was blocked by inhibition of the AMPK or ERK pathways through shRNA or via pharmacologic inhibitors of RAF (LY3009120), MEK (trametinib), or AMPK (compound C). The combination was found to be specific for RAS/RAF-mutant cells and had no significant effect in RAS/RAF-wild-type keratinocytes or melanoma cells. In vivo treatment of human xenografts in NSG mice with sorafenib and aspirin significantly reduced tumor volume compared with each single-agent treatment.Conclusions: Combination sorafenib and aspirin exerts cytotoxicity against RAS/RAF-mutant cells by simultaneously affecting two independent pathways and represents a promising novel strategy for the treatment of RAS-mutant cancers. Clin Cancer Res; 24(5); 1090-102. ©2017 AACR.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Aspirina/farmacologia , Neoplasias/tratamento farmacológico , Sorafenibe/farmacologia , Proteínas ras/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Aspirina/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Camundongos , Mutação , Neoplasias/genética , Proteínas Proto-Oncogênicas B-raf/genética , Transdução de Sinais/efeitos dos fármacos , Sorafenibe/uso terapêutico , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Invest Dermatol ; 138(4): 893-902, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29138054

RESUMO

Giant congenital nevi are associated with clinical complications such as neurocutaneous melanosis and melanoma. Virtually nothing is known about why some individuals develop these lesions. We previously identified the sonic hedgehog (Shh) pathway regulator Cdon as a candidate nevus modifier gene. Here we validate this by studying Cdon knockout mice, and go on to establishing the mechanism by which Shh exacerbates nevogenesis. Cdon knockout mice develop blue nevi without the need for somatic melanocyte oncogenic mutation. In a mouse model carrying melanocyte NRASQ61K, we found that strain backgrounds that carry genetic variants that cause increased keratinocyte Shh pathway activity, as measured by Gli1 and Gli2 expression, develop giant congenital nevi. Shh components are also active adjacent to human congenital nevi. Mechanistically, this exacerbation of nevogenesis is driven via the release of the melanocyte mitogen endothelin-1 from keratinocytes. We then suppressed nevus development in mice using Shh and endothelin antagonists. Our work suggests an aspect of nevus development whereby keratinocyte cytokines such as endothelin-1 can exacerbate nevogenesis, and provides potential therapeutic approaches for giant congenital nevi. Furthermore, it highlights the notion that germline genetic variation, in addition to somatic melanocyte mutation, can strongly influence the histopathological features of melanocytic nevi.


Assuntos
Endotelina-1/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Queratinócitos/metabolismo , Neoplasias Experimentais , Nevo Pigmentado/genética , Neoplasias Cutâneas/genética , Regulação para Cima , Animais , Feminino , Proteínas Hedgehog/biossíntese , Humanos , Queratinócitos/patologia , Masculino , Melanócitos/metabolismo , Melanócitos/patologia , Camundongos , Camundongos Knockout , Nevo Pigmentado/metabolismo , Nevo Pigmentado/patologia , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Ativação Transcricional , Células Tumorais Cultivadas
14.
Cell Cycle ; 17(5): 652-668, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28749250

RESUMO

We report for the first time the mechanism of action of the natural product thalicthuberine (TH) in prostate and cervical cancer cells. TH induced a strong accumulation of LNCaP cells in mitosis, severe mitotic spindle defects, and asymmetric cell divisions, ultimately leading to mitotic catastrophe accompanied by cell death through apoptosis. However, unlike microtubule-binding drugs (vinblastine and paclitaxel), TH did not directly inhibit tubulin polymerization when tested in a cell-free system, whereas it reduced cellular microtubule polymer mass in LNCaP cells. This suggests that TH indirectly targets microtubule dynamics through inhibition of a critical regulator or tubulin-associated protein. Furthermore, TH is not a major substrate for P-glycoprotein (Pgp), which is responsible for multidrug resistance in numerous cancers, providing a rationale to further study TH in cancers with Pgp-mediated treatment resistance. The identification of TH's molecular target in future studies will be of great value to the development of TH as potential treatment of multidrug-resistant tumors.


Assuntos
Alcaloides/farmacologia , Antimitóticos/farmacologia , Apoptose/efeitos dos fármacos , Fenantrenos/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/fisiologia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
15.
Mol Cancer Ther ; 16(9): 1934-1941, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28522591

RESUMO

The activity and efficacy of Aurora inhibitors have been reported in a wide range of cancer types. The most prominent Aurora inhibitor is alisertib, an investigational Aurora inhibitor that has been the subject of more than 30 clinical trials. Alisertib has inhibitory activity against both Aurora A and B, although it is considered to be primarily an Aurora A inhibitor in vivo Here, we show that alisertib inhibits both Aurora A and B in vivo in preclinical models of HPV-driven cervical cancer, and that it is the inhibition of Aurora A and B that provides the selectivity and efficacy of this drug in vivo in this disease setting. We also present formal evidence that alisertib requires progression through mitosis for its efficacy, and that it is unlikely to combine with drugs that promote a G2 DNA damage checkpoint response. This work demonstrates that inhibition of Aurora A and B is required for effective control of HPV-driven cancers by Aurora kinase inhibitors. Mol Cancer Ther; 16(9); 1934-41. ©2017 AACR.


Assuntos
Antineoplásicos/farmacologia , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase B/antagonistas & inibidores , Infecções por Papillomavirus/complicações , Inibidores de Proteínas Quinases/farmacologia , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/metabolismo , Animais , Azepinas/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Mitose/efeitos dos fármacos , Papillomaviridae , Pirimidinas/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Exp Dermatol ; 26(7): 649-655, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28109167

RESUMO

The advent of targeted therapies of metastatic melanoma, such as MAPK pathway inhibitors and immune checkpoint antagonists, has turned dermato-oncology from the "bad guy" to the "poster child" in oncology. Current targeted therapies are effective, although here is a clear need to develop combination therapies to delay the onset of resistance. Many antimelanoma drugs impact on the cell cycle but are also dependent on certain cell cycle phases resulting in cell cycle phase-specific drug insensitivity. Here, we raise the question: Have combination trials been abandoned prematurely as ineffective possibly only because drug scheduling was not optimized? Firstly, if both drugs of a combination hit targets in the same melanoma cell, cell cycle-mediated drug insensitivity should be taken into account when planning combination therapies, timing of dosing schedules and choice of drug therapies in solid tumors. Secondly, if the combination is designed to target different tumor cell subpopulations of a heterogeneous tumor, one drug effective in a particular subpopulation should not negatively impact on the other drug targeting another subpopulation. In addition to the role of cell cycle stage and progression on standard chemotherapeutics and targeted drugs, we discuss the utilization of cell cycle checkpoint control defects to enhance chemotherapeutic responses or as targets themselves. We propose that cell cycle-tailored targeting of metastatic melanoma could further improve therapy outcomes and that our real-time cell cycle imaging 3D melanoma spheroid model could be utilized as a tool to measure and design drug scheduling approaches.


Assuntos
Ciclo Celular , Melanoma/metabolismo , Melanoma/terapia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/terapia , Antineoplásicos/farmacologia , Terapia Combinada , Progressão da Doença , Sistemas de Liberação de Medicamentos , Humanos , Imageamento Tridimensional , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/uso terapêutico , Esferoides Celulares
17.
SLAS Discov ; 22(3): 298-308, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27872202

RESUMO

Malignant melanomas often arise from nevi, which result from initial oncogene-induced hyperproliferation of melanocytes that are maintained in a CDKN2A/p16-mediated senescent state. Thus, genes that can bypass this senescence barrier are likely to contribute to melanoma development. We have performed a gain-of-function screen of 17,030 lentivirally expressed human open reading frames (ORFs) in a melanoma cell line containing an inducible p16 construct to identify such genes. Genes known to bypass p16-induced senescence arrest, including the human papilloma virus 18 E7 gene ( HPV18E7), and genes such as the p16-binding CDK6 with expected functions, as well as panel of novel genes, were identified, including high-mobility group box (HMGB) proteins. A number of these were further validated in two other models of p16-induced senescence. Tissue immunohistochemistry demonstrated higher levels of CDK6 in primary melanomas compared with normal skin and nevi. Reduction of CDK6 levels drove melanoma cells expressing functional p16 into senescence, demonstrating its contribution to bypass senescence.


Assuntos
Pontos de Checagem do Ciclo Celular , Quinase 6 Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Regulação Neoplásica da Expressão Gênica , Ensaios de Triagem em Larga Escala , Melanócitos/metabolismo , Linhagem Celular Tumoral , Senescência Celular , Quinase 6 Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Biblioteca Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Genoma Humano , Células HEK293 , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Melanócitos/patologia , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Nevo/genética , Nevo/metabolismo , Nevo/patologia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Fases de Leitura Aberta , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia
18.
Mol Cancer Ther ; 16(1): 3-15, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27760837

RESUMO

The lack of a cure for metastatic castrate-resistant prostate cancer (mCRPC) highlights the urgent need for more efficient drugs to fight this disease. Here, we report the mechanism of action of the natural product 6α-acetoxyanopterine (6-AA) in prostate cancer cells. At low nanomolar doses, this potent cytotoxic alkaloid from the Australian endemic tree Anopterus macleayanus induced a strong accumulation of LNCaP and PC-3 (prostate cancer) cells as well as HeLa (cervical cancer) cells in mitosis, severe mitotic spindle defects, and asymmetric cell divisions, ultimately leading to mitotic catastrophe accompanied by cell death through apoptosis. DNA microarray of 6-AA-treated LNCaP cells combined with pathway analysis identified very similar transcriptional changes when compared with the anticancer drug vinblastine, which included pathways involved in mitosis, microtubule spindle organization, and microtubule binding. Like vinblastine, 6-AA inhibited microtubule polymerization in a cell-free system and reduced cellular microtubule polymer mass. Yet, microtubule alterations that are associated with resistance to microtubule-destabilizing drugs like vinca alkaloids (vinblastine/vincristine) or 2-methoxyestradiol did not confer resistance to 6-AA, suggesting a different mechanism of microtubule interaction. 6-AA is a first-in-class microtubule inhibitor that features the unique anopterine scaffold. This study provides a strong rationale to further develop this novel structure class of microtubule inhibitor for the treatment of malignant disease. Mol Cancer Ther; 16(1); 3-15. ©2016 AACR.


Assuntos
Antimitóticos/farmacologia , Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Moduladores de Tubulina/farmacologia , Antimitóticos/química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Produtos Biológicos/química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Masculino , Mitose/genética , Neoplasias da Próstata/metabolismo , Multimerização Proteica/efeitos dos fármacos , Fuso Acromático/efeitos dos fármacos , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Vimblastina/farmacologia
19.
Curr Med Chem ; 24(15): 1504-1519, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27919216

RESUMO

Interactions between the decatenation checkpoint and Topoisomerase II (TopoII) are vital for maintaining integrity of the genome. Agents that target this enzyme have been in clinical use in cancer therapy for over 30 years with great success. The types of compounds that have been developed to target TopoII are broadly divided into poisons and catalytic inhibitors. The TopoII poisons are in clinical use as anti-cancer therapies, although in common to most chemotherapeutic agents, they display considerable normal tissue toxicity. Inhibition of the TopoIIb isoform has been implicated in this cytotoxicity. Response to TopoII active agents is determined by several factors, but cell cycle checkpoints play a large role in sensitivity and resistance. The G2/M phase checkpoints are of particular importance in considering the effectiveness of these drugs and are reviewed in this article. Functionality of the ATM dependent decatenation checkpoint may represent a new avenue for selective cancer therapy. Here we review the function of TopoII, the anti-cancer mechanisms and limitations of current catalytic inhibitors and poisons, and their influence on cell cycle checkpoints. We will also assess potential new mechanisms for targeting this enzyme to limit normal tissue toxicity, and how the cell cycle checkpoint triggered by these drugs may provide an alternative and possibly better target for novel therapies.


Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Inibidores da Topoisomerase II/toxicidade , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , DNA Topoisomerases Tipo II/química , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/uso terapêutico , Proteína Supressora de Tumor p53/metabolismo
20.
Oncotarget ; 7(38): 61000-61020, 2016 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-27876705

RESUMO

Epithelial to mesenchymal transition (EMT) is a developmental program that has been implicated in progression, metastasis and therapeutic resistance of some carcinomas. To identify genes whose overexpression drives EMT, we screened a lentiviral expression library of 17000 human open reading frames (ORFs) using high-content imaging to quantitate cytoplasmic vimentin. Hits capable of increasing vimentin in the mammary carcinoma-derived cell line MDA-MB-468 were confirmed in the non-tumorigenic breast-epithelial cell line MCF10A. When overexpressed in this model, they increased the rate of cell invasion through Matrigel™, induced mesenchymal marker expression and reduced expression of the epithelial marker E-cadherin. In gene-expression datasets derived from breast cancer patients, the expression of several novel genes correlated with expression of known EMT marker genes, indicating their in vivo relevance. As EMT-associated properties are thought to contribute in several ways to cancer progression, genes identified in this study may represent novel targets for anti-cancer therapy.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal , Genoma Humano , Antígenos CD , Caderinas/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Lentivirus/metabolismo , Fases de Leitura Aberta , Plasmídeos/metabolismo , Vimentina/metabolismo
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