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1.
Transl Oncol ; 14(12): 101229, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34592589

RESUMO

Tumour metastasis accounts for over 90% of cancer related deaths. The platelet is a key blood component, which facilitates efficient metastasis. This study aimed to understand the molecular mechanisms involved in tumour-platelet cell interactions. The interaction between cancer cells and platelets was examined in 15 epithelial cell lines, representing 7 cancer types. Gene expression analysis of EMT-associated and cancer stemness genes was performed by RT-PCR. Whole transcriptome analysis (WTA) was performed using Affymetrix 2.0ST arrays on a platelet co-cultured ovarian model. Platelet adhesion and activation occurred across all tumour types. WTA identified increases in cellular movement, migration, invasion, adhesion, development, differentiation and inflammation genes and decreases in processes associated with cell death and survival following platelet interaction. Increased invasive capacity was also observed in a subset of cell lines. A cross-comparison with a platelet co-cultured mouse model identified 5 common altered genes; PAI-1, PLEK2, CD73, TNC, and SDPR. Platelet cancer cell interactions are a key factor in driving the pro-metastatic phenotype and appear to be mediated by 5 key genes which have established roles in metastasis. Targeting these metastasis mediators could improve cancer patient outcomes.

2.
PLoS One ; 15(12): e0243715, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33370338

RESUMO

Despite the use of front-line anticancer drugs such as paclitaxel for ovarian cancer treatment, mortality rates have remained almost unchanged for the past three decades and the majority of patients will develop recurrent chemoresistant disease which remains largely untreatable. Overcoming chemoresistance or preventing its onset in the first instance remains one of the major challenges for ovarian cancer research. In this study, we demonstrate a key link between senescence and inflammation and how this complex network involving the biomarkers MAD2, TLR4 and MyD88 drives paclitaxel resistance in ovarian cancer. This was investigated using siRNA knockdown of MAD2, TLR4 and MyD88 in two ovarian cancer cell lines, A2780 and SKOV-3 cells and overexpression of MyD88 in A2780 cells. Interestingly, siRNA knockdown of MAD2 led to a significant increase in TLR4 gene expression, this was coupled with the development of a highly paclitaxel-resistant cell phenotype. Additionally, siRNA knockdown of MAD2 or TLR4 in the serous ovarian cell model OVCAR-3 resulted in a significant increase in TLR4 or MAD2 expression respectively. Microarray analysis of SKOV-3 cells following knockdown of TLR4 or MAD2 highlighted a number of significantly altered biological processes including EMT, complement, coagulation, proliferation and survival, ECM remodelling, olfactory receptor signalling, ErbB signalling, DNA packaging, Insulin-like growth factor signalling, ion transport and alteration of components of the cytoskeleton. Cross comparison of the microarray data sets identified 7 overlapping genes including MMP13, ACTBL2, AMTN, PLXDC2, LYZL1, CCBE1 and CKS2. These results demonstrate an important link between these biomarkers, which to our knowledge has never before been shown in ovarian cancer. In the future, we hope that triaging patients into alterative treatment groups based on the expression of these three biomarkers or therapeutic targeting of the mechanisms they are involved in will lead to improvements in patient outcome and prevent the development of chemoresistance.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Biomarcadores Tumorais/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Linhagem Celular Tumoral , Senescência Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteínas Mad2/genética , Fator 88 de Diferenciação Mieloide/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Paclitaxel/uso terapêutico , RNA Interferente Pequeno/metabolismo , Receptor 4 Toll-Like/genética
3.
Cancer Lett ; 469: 11-21, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31593803

RESUMO

MAD2 is an intriguing protein, which has been associated with poor survival in cancer. Depending on the organ-specific cancer, either high expression or low expression levels have been correlated with low survival rates in patients. MAD2 is also a marker of contradiction. The normal function of MAD2 is to accumulate at kinetochores and generate a wait signal preventing the cell from progressing to anaphase of the cell cycle until the spindle microtubules have correctly aligned with the kinetochores on each chromosome. This process ensures that sister chromatids segregate correctly into each new daughter cell upon cellular division. Thus, the correct function of MAD2 and this crucial cell cycle checkpoint, the spindle assembly checkpoint (SAC), is essential for faithful replicative cell division, the prevention of chromosomal abnormalities and the development of cancer. Surprisingly when MAD2 is supressed for example through siRNA, this results in the induction of cellular senescence or cell cycle arrest. This is an inherent contradiction as normally the dispersement of MAD2 would signal to a cell that they should proceed to anaphase as spindle microtubules have correctly aligned with each chromatid for cell division. In the inverse setting; a second contradiction, high MAD2 expression in cancer patients generally correlates with abnormal chromosome number. However, in normal cells high expression of MAD2 would limit this by generating a wait signal to prevent the cell from proceeding through the cell cycle. In this review article we aim to make sense of the MADness and review the current knowledge of MAD2 and its role in cancer.


Assuntos
Aberrações Cromossômicas , Regulação Neoplásica da Expressão Gênica , Pontos de Checagem da Fase M do Ciclo Celular/genética , Proteínas Mad2/metabolismo , Neoplasias/genética , Animais , Hipóxia Celular/genética , Senescência Celular/genética , Modelos Animais de Doenças , Humanos , Neoplasias/mortalidade , Neoplasias/patologia , Prognóstico , Fuso Acromático/genética , Fuso Acromático/patologia , Regulação para Cima
4.
Oncotarget ; 6(35): 37919-29, 2015 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-26473288

RESUMO

Prostate cancer continues to be a major cause of morbidity and mortality in men, but a method for accurate prognosis in these patients is yet to be developed. The recent discovery of altered endosomal biogenesis in prostate cancer has identified a fundamental change in the cell biology of this cancer, which holds great promise for the identification of novel biomarkers that can predict disease outcomes. Here we have identified significantly altered expression of endosomal genes in prostate cancer compared to non-malignant tissue in mRNA microarrays and confirmed these findings by qRT-PCR on fresh-frozen tissue. Importantly, we identified endosomal gene expression patterns that were predictive of patient outcomes. Two endosomal tri-gene signatures were identified from a previously published microarray cohort and had a significant capacity to stratify patient outcomes. The expression of APPL1, RAB5A, EEA1, PDCD6IP, NOX4 and SORT1 were altered in malignant patient tissue, when compared to indolent and normal prostate tissue. These findings support the initiation of a case-control study using larger cohorts of prostate tissue, with documented patient outcomes, to determine if different combinations of these new biomarkers can accurately predict disease status and clinical progression in prostate cancer patients.


Assuntos
Biomarcadores Tumorais/genética , Endossomos/metabolismo , Recidiva Local de Neoplasia/genética , Neoplasia Prostática Intraepitelial/genética , Neoplasias da Próstata/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Estudos de Coortes , Progressão da Doença , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Seguimentos , Perfilação da Expressão Gênica , Humanos , Masculino , NADPH Oxidase 4 , NADPH Oxidases/genética , Gradação de Tumores , Metástase Neoplásica , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Prognóstico , Neoplasia Prostática Intraepitelial/mortalidade , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Proteínas de Transporte Vesicular/genética , Proteínas rab5 de Ligação ao GTP/genética
5.
BMC Cancer ; 15: 627, 2015 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-26353776

RESUMO

BACKGROUND: Platelet-cancer cell interactions play a key role in successful haematogenous metastasis. Disseminated malignancy is the leading cause of death among ovarian cancer patients. It is unknown why different ovarian cancers have different metastatic phenotypes. To investigate if platelet-cancer cell interactions play a role, we characterized the response of ovarian cancer cell lines to platelets both functionally and at a molecular level. METHODS: Cell lines 59 M and SK-OV-3 were used as in vitro model systems of metastatic ovarian cancer. Platelet cloaking of each cell line was quantified by flow cytometry. Matrigel invasion chamber assays were used to assess the invasive capacity of the cell lines. The induction of an EMT was assessed by morphology analysis and by gene expression analysis of a panel of 11 EMT markers using TaqMan RT-PCR. RESULTS: SK-OV-3 cells adhered to and activated more platelets than 59 M cells (p = 0.0333). Platelets significantly promoted the ability of only SK-OV-3 cells to invade (p ≤ 0.0001). Morphology and transcritpome analysis indicated that platelets induce an epithelial-to-mesenchymal transition phenotype in both cells lines, with a more exaggerated response in SK-OV-3 cells. Next, we investigated if antiplatelet agents could abrogate the platelet-induced aggressive phenotype in SK-OV-3 cells. Both aspirin (p ≤ 0.05) and 2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate (P2Y12 inhibitor; p ≤ 0.01) significantly decreased their invasion capacity, and effectively reverted invasion to levels comparable to SK-OV-3 cells alone. CONCLUSION: While there is increasing evidence for the cancer-protective effect of aspirin, this study suggests P2Y12 inhibition may also play a role. Understanding these complex interactions between platelets and cancer cells could ultimately allow the establishment of therapies tailored to inhibiting metastasis, thus significantly reducing cancer morbidity.


Assuntos
Aspirina/farmacologia , Plaquetas/fisiologia , Invasividade Neoplásica , Neoplasias Ovarianas/patologia , Inibidores da Agregação Plaquetária/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y12/efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Humanos , Metástase Neoplásica , Reação em Cadeia da Polimerase em Tempo Real
6.
BMC Cancer ; 15: 547, 2015 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-26205780

RESUMO

BACKGROUND: Ovarian cancer is associated with poor long-term survival due to late diagnosis and development of chemoresistance. Tumour hypoxia is associated with many features of tumour aggressiveness including increased cellular proliferation, inhibition of apoptosis, increased invasion and metastasis, and chemoresistance, mostly mediated through hypoxia-inducible factor (HIF)-1α. While HIF-1α has been associated with platinum resistance in a variety of cancers, including ovarian, relatively little is known about the importance of the duration of hypoxia. Similarly, the gene pathways activated in ovarian cancer which cause chemoresistance as a result of hypoxia are poorly understood. This study aimed to firstly investigate the effect of hypoxia duration on resistance to cisplatin in an ovarian cancer chemoresistance cell line model and to identify genes whose expression was associated with hypoxia-induced chemoresistance. METHODS: Cisplatin-sensitive (A2780) and cisplatin-resistant (A2780cis) ovarian cancer cell lines were exposed to various combinations of hypoxia and/or chemotherapeutic drugs as part of a 'hypoxia matrix' designed to cover clinically relevant scenarios in terms of tumour hypoxia. Response to cisplatin was measured by the MTT assay. RNA was extracted from cells treated as part of the hypoxia matrix and interrogated on Affymetrix Human Gene ST 1.0 arrays. Differential gene expression analysis was performed for cells exposed to hypoxia and/or cisplatin. From this, four potential markers of chemoresistance were selected for evaluation in a cohort of ovarian tumour samples by RT-PCR. RESULTS: Hypoxia increased resistance to cisplatin in A2780 and A2780cis cells. A plethora of genes were differentially expressed in cells exposed to hypoxia and cisplatin which could be associated with chemoresistance. In ovarian tumour samples, we found trends for upregulation of ANGPTL4 in partial responders and down-regulation in non-responders compared with responders to chemotherapy; down-regulation of HER3 in partial and non-responders compared to responders; and down-regulation of HIF-1α in non-responders compared with responders. CONCLUSION: This study has further characterized the relationship between hypoxia and chemoresistance in an ovarian cancer model. We have also identified many potential biomarkers of hypoxia and platinum resistance and provided an initial validation of a subset of these markers in ovarian cancer tissues.


Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Angiopoietinas/genética , Biomarcadores Tumorais/metabolismo , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Ovarianas/patologia , Receptor ErbB-3/genética
7.
J Clin Pathol ; 68(9): 692-702, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26038242

RESUMO

AIMS: Targeting the stem cell properties of tumor-initiating cells is an avenue through which cancer treatment may be improved. Before this can be achieved, so-called 'cancer stem cell' (CSC) models must be developed and characterized in specific malignancies. METHODS: In this study, holoclone formation assays were used to characterise stem-like molecular signatures in prostate cancer (PCa) cells. RESULTS: LNCaP and PC3 parent cells were capable of responding to stem cell differentiation morphogen retinoic acid (RA), suggesting the presence of inherent stem-like properties. LNCaP cells, which represent early, androgen-responsive disease, formed holoclones after twenty six days. PC3 cells, which represent advanced, metastatic, castration-resistant disease, formed holoclones after only six days. Holoclones displayed decreased expression of RA-genes, suggesting a more immature, less differentiated phenotype. Gene and microRNA arrays demonstrated that holoclones downregulated a number of stem cell differentiation regulators while displaying enhanced regulation of G2 to M transition and the mitotic spindle checkpoint components of the cell cycle. PC3 holoclones displayed pronounced downregulation of known regulators of osteoblast differentiation from mesenchymal stem cells and Epithelial Mesenchymal Transition. CONCLUSIONS: Our results suggest that some PCa cells retain the ability to transition to a more immature state in which differentiation and metastatic mechanisms are suppressed. The highlighting of osteoblast differentiation regulators in this mechanism is particularly notable, considering the propensity of PCa to metastasise to bone.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Neoplásicas/patologia , Osteoblastos/citologia , Neoplasias da Próstata/patologia , Transcriptoma , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Masculino , Células-Tronco Neoplásicas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo
8.
PLoS One ; 9(6): e100816, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24977712

RESUMO

The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.


Assuntos
Cistadenocarcinoma Seroso/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Fator 88 de Diferenciação Mieloide/genética , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Receptor 4 Toll-Like/genética , Idoso , Antineoplásicos Fitogênicos/farmacologia , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Cistadenocarcinoma Seroso/diagnóstico , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/mortalidade , Feminino , Genótipo , Humanos , Imuno-Histoquímica , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Fator 88 de Diferenciação Mieloide/metabolismo , Neoplasias Epiteliais e Glandulares/diagnóstico , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/mortalidade , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/mortalidade , Paclitaxel/farmacologia , Fenótipo , Prognóstico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/metabolismo
9.
J Ovarian Res ; 5(1): 2, 2012 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-22260314

RESUMO

BACKGROUND: Malignant ovarian disease is characterised by high rates of mortality due to high rates of recurrent chemoresistant disease. Anecdotal evidence indicates this may be due to chemoresistant properties of cancer stem cells (CSCs). However, our understanding of the role of CSCs in recurrent ovarian disease remains sparse. In this study we used gene microarrays and meta-analysis of our previously published microRNA (miRNA) data to assess the involvement of cancer stemness signatures in recurrent ovarian disease. METHODS: Microarray analysis was used to characterise early regulation events in an embryonal carcinoma (EC) model of cancer stemness. This was then compared to our previously published microarray data from a study of primary versus recurrent ovarian disease. In parallel, meta-analysis was used to identify cancer stemness miRNA signatures in tumor patient samples. RESULTS: Microarray analysis demonstrated a 90% difference between gene expression events involved in early regulation of differentiation in murine EC (mEC) and embryonic stem (mES) cells. This contrasts the known parallels between mEC and mES cells in the undifferentiated and well-differentiated states. Genelist comparisons identified a cancer stemness signature set of genes in primary versus recurrent data, a subset of which are known p53-p21 regulators. This signature is present in primary and recurrent or in primary alone but essentially never in recurrent tumors specifically. Meta-analysis of miRNA expression showed a much stronger cancer stemness signature within tumor samples. This miRNA signature again related to p53-p21 regulation and was expressed prominently in recurrent tumors. Our data indicate that the regulation of p53-p21 in ovarian cancer involves, at least partially, a cancer stemness component. CONCLUSION: We present a p53-p21 cancer stemness signature model for ovarian cancer. We propose that this may, at least partially, differentially regulate the p53-p21 mechanism in ovarian disease. Targeting CSCs within ovarian cancer represents a potential therapeutic avenue.

10.
Lab Chip ; 11(14): 2447-54, 2011 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-21655638

RESUMO

Cell sorting and separation techniques are essential tools for cell biology research and for many diagnostic and therapeutic applications. For many of these applications, it is imperative that heterogeneous populations of cells are segregated according to their cell type and that individual cells can be isolated and analysed. We present a novel technique to isolate single cells encapsulated in a picolitre sized droplet that are then deposited by inkjet-like printing at defined locations for downstream genomic analysis. The single-cell-manipulator (SCM) developed for this purpose consists of a dispenser chip to print cells contained in a free flying droplet, a computer vision system to detect single-cells inside the dispenser chip prior to printing, and appropriate automation equipment to print single-cells onto defined locations on a substrate. This technique is spatially dynamic, enabling cell printing on a wide range of commonly used substrates such as microscope slides, membranes and microtiter plates. Demonstration experiments performed using the SCM resulted in a printing efficiency of 87% for polystyrene microbeads of 10 µm size. When the SCM was applied to a cervical cancer cell line (HeLa), a printing efficiency of 87% was observed and a post-SCM cell viability rate of 75% was achieved.


Assuntos
Separação Celular/métodos , Algoritmos , Separação Celular/instrumentação , Sobrevivência Celular , Processamento Eletrônico de Dados , Células HeLa , Humanos
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