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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.
Breast Cancer Res Treat ; 181(3): 571-580, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32378053

RESUMO

PURPOSE: The association between pathological complete response (pCR) in patients receiving neoadjuvant chemotherapy (NAC) for breast cancer and Circulating Tumour Cells (CTCs) is not clear. The aim of this study was to assess whether CTC enumeration could be used to predict pathological response to NAC in breast cancer as measured by the Miller-Payne grading system. METHODS: Twenty-six patients were recruited, and blood samples were taken pre- and post-NAC. CTCs were isolated using the ScreenCell device and stained using a modified Giemsa stain. CTCs were enumerated by 2 pathologists and classified as single CTCs, doublets, clusters/microemboli and correlated with the pathological response as measured by the Miller-Payne grading system. χ2 or ANOVA was performed in SPSS 24.0 statistics software for associations. RESULTS: 89% of patients had invasive ductal carcinoma (IDC) and 11% invasive lobular carcinoma (ILC). At baseline 85% of patients had CTCs present, median 7 (0-161) CTCs per 3 ml of whole blood. Post-chemotherapy, 58% had an increase in CTCs. This did not correlate with the Miller-Payne grade of response. No significant association was identified between the number of CTCs and clinical characteristics; however, we did observe a correlation between pre-treatment CTC counts and body mass index, p < 0.05. CONCLUSIONS: Patients with a complete response to NAC still had CTCs present, suggesting enumeration is not sufficient to aid surgery stratification. Additional characterisation and larger studies are needed to further characterise CTCs isolated pre- and post-chemotherapy. Long-term follow-up of these patients will determine the significance of CTCs in NAC breast cancer patients.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/patologia , Terapia Neoadjuvante/mortalidade , Células Neoplásicas Circulantes/patologia , Adulto , Idoso , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/tratamento farmacológico , Carcinoma Lobular/metabolismo , Estudos de Coortes , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Células Neoplásicas Circulantes/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Taxa de Sobrevida
4.
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
5.
PLoS One ; 14(3): e0211538, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30908480

RESUMO

Tumour cell immune evasion is a principal hallmark of successful metastasis. Tumour cells in the vasculature adopt a platelet cloak that efficiently suppresses the innate immune system by directly inhibiting Natural Killer (NK) cells, which normally function to neutralise spreading cancers. Here we describe two novel mechanisms of tumour cell evasion of NK cell anti-tumour functions. The first, an 'immune decoy' mechanism in which platelets induce the release of soluble NKG2D ligands from the tumour cell to mask detection and actively suppress NK cell degranulation and inflammatory cytokine (IFNγ) production, concomitantly. This represents a double-hit to immune clearance of malignant cells during metastasis. The second mechanism, a platelet-derived TGFß-mediated suppression of the CD226/CD96-CD112/CD155 axis, is a novel pathway with poorly understood anti-cancer functions. We have demonstrated that platelets robustly suppress surface expression of CD226 and CD96 on the NK cell surface and their associated ligands on the tumour cell to further enhance NK cell suppression. These highly evolved mechanisms promote successful tumour immune evasion during metastasis and provide a unique opportunity for studying the complexity of cellular interactions in the metastatic cascade and thus novel targets for cancer immunotherapy.


Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Plaquetas/imunologia , Células Matadoras Naturais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Metástase Neoplásica/imunologia , Evasão Tumoral , Antígenos CD/metabolismo , Linhagem Celular Tumoral , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunidade Inata , Interferon gama/metabolismo , Nectinas/metabolismo , Receptores Virais/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo
6.
Cell Death Dis ; 8(10): e3128, 2017 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-29048400

RESUMO

It is long established that tumour-initiating cancer stem cells (CSCs) possess chemoresistant properties. However, little is known of the mechanisms involved, particularly with respect to the organisation of CSCs as stem-progenitor-differentiated cell hierarchies. Here we aimed to elucidate the relationship between CSC hierarchies and chemoresistance in an ovarian cancer model. Using a single cell-based approach to CSC discovery and validation, we report a novel, four-component CSC hierarchy based around the markers cluster of differentiation 10 (CD10) and aldehyde dehydrogenase (ALDH). In a change to our understanding of CSC biology, resistance to chemotherapy drug cisplatin was found to be the sole property of CD10-/ALDH- CSCs, while all four CSC types were sensitive to chemotherapy drug paclitaxel. Cisplatin treatment quickly altered the hierarchy, resulting in a three-component hierarchy dominated by the cisplatin-resistant CD10-/ALDH- CSC. This organisation was found to be hard-wired in a long-term cisplatin-adapted model, where again CD10-/ALDH- CSCs were the sole cisplatin-resistant component, and all CSC types remained paclitaxel-sensitive. Molecular analysis indicated that cisplatin resistance is associated with inherent- and adaptive-specific drug efflux and DNA-damage repair mechanisms. Clinically, low CD10 expression was consistent with a specific set of ovarian cancer patient samples. Collectively, these data advance our understanding of the relationship between CSC hierarchies and chemoresistance, which was shown to be CSC- and drug-type specific, and facilitated by specific and synergistic inherent and adaptive mechanisms. Furthermore, our data indicate that primary stage targeting of CD10-/ALDH- CSCs in specific ovarian cancer patients in future may facilitate targeting of recurrent disease, before it ever develops.


Assuntos
Aldeído Desidrogenase/genética , Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Células-Tronco Neoplásicas/patologia , Neprilisina/genética , Neoplasias Ovarianas/patologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Dano ao DNA , Reparo do DNA , Feminino , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/tratamento farmacológico
7.
Cell Death Differ ; 24(11): 1975-1986, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28885616

RESUMO

We have previously reported that myeloid differentiation primary response gene 88 (MyD88) is downregulated during all-trans retinoic acid (RA)-induced differentiation of pluripotent NTera2 human embryonal carcinoma cells (hECCs), whereas its maintained expression is associated with RA differentiation resistance in nullipotent 2102Ep hECCs. MyD88 is the main adapter for toll-like receptor (TLR) signalling, where it determines the secretion of chemokines and cytokines in response to pathogens. In this study, we report that loss of MyD88 is essential for RA-facilitated differentiation of hECCs. Functional analysis using a specific MyD88 peptide inhibitor (PepInh) demonstrated that high MyD88 expression in the self-renewal state inhibits the expression of a specific set of HOX genes. In NTera2 cells, MyD88 is downregulated during RA-induced differentiation, a mechanism that could be broadly replicated by MyD88 PepInh treatment of 2102Ep cells. Notably, MyD88 inhibition transitioned 2102Ep cells into a stable, self-renewing state that appears to be primed for differentiation upon addition of RA. At a molecular level, MyD88 inhibition combined with RA treatment upregulated HOX, RA signalling and TLR signalling genes. These events permit differentiation through a standard downregulation of Oct4-Sox2-Nanog mechanism. In line with its role in regulating secretion of specific proteins, conditioned media experiments demonstrated that differentiated (MyD88 low) NTera2 cell media was sufficient to differentiate NTera2 cells. Protein array analysis indicated that this was owing to secretion of factors known to regulate angiogenesis, neurogenesis and all three branches of TGF-ß Superfamily signalling. Collectively, these data offer new insights into RA controlled differentiation of pluripotent cells, with notable parallels to the ground state model of embryonic stem cell self-renewal. These data may provide insights to facilitate improved differentiation protocols for regenerative medicine and differentiation-therapies in cancer treatment.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco de Carcinoma Embrionário/patologia , Fator 88 de Diferenciação Mieloide/metabolismo , Células-Tronco Pluripotentes/patologia , Tretinoína/farmacologia , Diferenciação Celular/genética , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Células-Tronco de Carcinoma Embrionário/efeitos dos fármacos , Células-Tronco de Carcinoma Embrionário/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Mesoderma/patologia , Modelos Biológicos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
8.
Int J Mol Med ; 38(2): 433-45, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27353001

RESUMO

Thyroid cancer is the most common endocrine malignancy and accounts for the majority of endocrine cancer-related deaths each year. Our group and others have previously demonstrated dysfunctional microRNA (miRNA or miR) expression in the context of thyroid cancer. The objective of the present study was to investigate the impact of synthetic manipulation of expression of miR-25 and miR-222 in benign and malignant thyroid cells. miR-25 and miR-222 expression was upregulated in 8505C (an anaplastic thyroid cell line) and Nthy-ori (a SV40-immortalised thyroid cell line) cells, respectively. A transcriptomics-based approach was utilised to identify targets of the two miRNAs and real-time PCR and western blotting were used to validate a subset of the targets. Almost 100 mRNAs of diverse functions were found to be either directly or indirectly targeted by both miR-222 and miR-25 [fold change ≥2, false discovery rate (FDR) ≤0.05]. Gene ontology analysis showed the miR-25 gene target list to be significantly enriched for genes involved in cell adhesion. Fluidigm real-time PCR technologies were used to validate the downregulation of 23 and 22 genes in response to miR-25 and miR-222 overexpression, respectively. The reduction of the expression of two miR-25 protein targets, TNF-related apoptosis­inducing ligand (TRAIL) and mitogen-activated protein kinase kinase 4 (MEK4), was also validated. Manipulating the expression of both miR-222 and miR-25 influenced diverse gene expression changes in thyroid cells. Increased expression of miR-25 reduced MEK4 and TRAIL protein expression, and cell adhesion and apoptosis are important aspects of miR-25 functioning in thyroid cells.


Assuntos
Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , MAP Quinase Quinase 4/metabolismo , MicroRNAs/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Carcinoma Anaplásico da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/patologia , Regulação para Baixo/genética , Humanos , MAP Quinase Quinase 4/genética , MicroRNAs/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia
9.
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
10.
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
11.
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
12.
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
13.
Cancer Lett ; 356(2 Pt B): 628-36, 2015 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-25451316

RESUMO

Ovarian cancer is the seventh most common cancer in women and the most frequent cause of gynaecological malignancy-related mortality in women. Currently, no standardized reliable screening test exists. MicroRNA profiling has allowed the identification of signatures associated with diagnosis, prognosis and response to treatment of human tumours. The aim of this study was to determine if a microRNA signature could distinguish between malignant and benign ovarian disease. A training set of 5 serous ovarian carcinomas and 5 benign serous cystadenomas were selected for the initial experiments. The validation set included 20 serous ovarian carcinomas and 20 benign serous cystadenomas. The serum/plasma focus microRNA Exiqon panel was used for the training set. For the validation set a pick and mix Exiqon panel, which focuses on microRNAs of interest was used. A panel of 4 microRNAs (let-7i-5p, miR-122, miR-152-5p and miR-25-3p) was significantly down regulated in cancer patients. These microRNAs target WNT signalling, AKT/mTOR and TLR-4/MyD88, which have previously been found to play a role in ovarian carcinogenesis and chemoresistance. let-7i-5p, miR-122, miR-152-5p and miR-25-3p could act as diagnostic biomarkers in ovarian cancer.


Assuntos
Biomarcadores Tumorais/sangue , Cistadenocarcinoma Seroso/diagnóstico , MicroRNAs/sangue , MicroRNAs/genética , Neoplasias Ovarianas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Cistadenocarcinoma Seroso/sangue , Cistadenocarcinoma Seroso/genética , Feminino , Seguimentos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Hemólise , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/genética , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
BMC Genomics ; 15: 711, 2014 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-25156079

RESUMO

BACKGROUND: SOX2 is a core component of the transcriptional network responsible for maintaining embryonal carcinoma cells (ECCs) in a pluripotent, undifferentiated state of self-renewal. As such, SOX2 is an oncogenic transcription factor and crucial cancer stem cell (CSC) biomarker in embryonal carcinoma and, as more recently found, in the stem-like cancer cell component of many other malignancies. SOX2 is furthermore a crucial factor in the maintenance of adult stem cell phenotypes and has additional roles in cell fate determination. The SOX2-linked microRNA (miRNA) transcriptome and regulome has not yet been fully defined in human pluripotent cells or CSCs. To improve our understanding of the SOX2-linked miRNA regulatory network as a contribution to the phenotype of these cell types, we used high-throughput differential miRNA and gene expression analysis combined with existing genome-wide SOX2 chromatin immunoprecipitation (ChIP) data to map the SOX2 miRNA transcriptome in two human embryonal carcinoma cell (hECC) lines. RESULTS: Whole-microRNAome and genome analysis of SOX2-silenced hECCs revealed many miRNAs regulated by SOX2, including several with highly characterised functions in both cancer and embryonic stem cell (ESC) biology. We subsequently performed genome-wide differential expression analysis and applied a Monte Carlo simulation algorithm and target prediction to identify a SOX2-linked miRNA regulome, which was strongly enriched with epithelial-to-mesenchymal transition (EMT) markers. Additionally, several deregulated miRNAs important to EMT processes had SOX2 binding sites in their promoter regions. CONCLUSION: In ESC-like CSCs, SOX2 regulates a large miRNA network that regulates and interlinks the expression of crucial genes involved in EMT.


Assuntos
Células-Tronco Embrionárias/metabolismo , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXB1/genética , Sítios de Ligação , Linhagem Celular , Transformação Celular Neoplásica/genética , Desenvolvimento Embrionário/genética , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Inativação Gênica , Humanos , Neoplasias/genética , Fenótipo , Regiões Promotoras Genéticas , Ligação Proteica
15.
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
16.
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.

17.
PLoS One ; 6(10): e26125, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22022533

RESUMO

Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.


Assuntos
Degranulação Celular , Neovascularização Patológica/patologia , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/patologia , Adesividade Plaquetária , Transdução de Sinais , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Apirase/farmacologia , Ácido Araquidônico/farmacologia , Degranulação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Fragmentos de Peptídeos/farmacologia , Adesividade Plaquetária/efeitos dos fármacos , Receptores Purinérgicos P2Y1/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Receptores de Trombina/antagonistas & inibidores , Receptores de Trombina/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos
18.
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
19.
Int J Gynecol Cancer ; 21(2): 206-12, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21270603

RESUMO

INTRODUCTION: Cervical cancer is the second most common cancer affecting women worldwide. It is characterized by chromosomal aberrations and alteration in the expression levels of many cell cycle regulatory proteins. MYBL2 is a member of the MYB proto-oncogene family that encodes DNA binding proteins. These proteins are involved in cell proliferation and control of cellular differentiation. MATERIALS AND METHODS: Four established cervical cancer cell lines were examined and compared with normal cervix using gene expression profiling and comparative genomic hybridization, and results were correlated to identify potential novel cervical cancer biomarkers. Results were validated using TaqMan polymerase chain reaction, and the potential role of MYBL2 as a clinical biomarker was then evaluated by immunohistochemistry on 30 tissue samples. RESULTS: MYBL2 was found to be overexpressed in the cervical cancer cell lines by gene expression profiling, and this result was confirmed using TaqMan polymerase chain reaction. Analysis of comparative genomic hybridization data indicated that chromosome 20q13.1, which encodes the MYBL2 gene, was amplified in the human papillomavirus (HPV) type 16-positive CaSki and SiHa cell lines but not in the HPV-18-positive HeLa or HPV-negative C33A cell lines. DISCUSSION: Although MYBL2 staining was predominantly absent in normal cervical epithelium, strong staining (score of 2 or 3) was identified in all cases of cervical intraepithelial neoplasia, cervical glandular intraepithelial neoplasia, and invasive cancer on immunohistochemistry. In addition, strong staining of a population of diffusely scattered single cells is identified. We postulate that these may represent so-called cancer stem-like cells.


Assuntos
Biomarcadores Tumorais/genética , Proteínas de Ciclo Celular/genética , Transativadores/genética , Neoplasias do Colo do Útero/genética , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias do Colo do Útero/patologia
20.
Analyst ; 135(12): 3087-93, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20967345

RESUMO

The main aetiology of cervical cancer is infection with high-risk human papillomavirus (HPV). Cervical cancer is almost 100% curable if detected in the early stages. Thus, information about the presence and levels of HPV in patient samples has high clinical value. As current screening methods, such as the Pap smear test, are highly subjective and in many cases show low sensitivity and specificity, new supportive techniques are desirable to improve the quality of cervical cancer screening. In this study, vibrational spectroscopic techniques (Raman and Fourier Transform Infra Red absorption) have been applied to the investigation of four cervical cancer cell lines: HPV negative C33A, HPV-18 positive HeLa with 20-50 integrated HPV copies per cell, HPV-16 positive SiHa with 1-2 integrated HPV strands per cell and HPV-16 positive CaSki containing 60-600 integrated HPV copies per cell. Results show that vibrational spectroscopic techniques can discriminate between the cell lines and elucidate cellular differences originating from proteins, nucleic acids and lipids. Similarities between C33A and SiHa cells were exhibited in the Raman and infrared spectra and were confirmed by Principal Component Analysis (PCA). Analysis of the biochemical composition of the investigated cells, with the aid of PCA, showed a clear discrimination between the C33A-SiHa group and HeLa and CaSki cell lines indicating the potential of vibrational spectroscopic techniques as a support to current methods for cervical cancer screening.


Assuntos
Papillomavirus Humano 18/patogenicidade , Infecções por Papillomavirus/complicações , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Análise Espectral Raman/métodos , Neoplasias do Colo do Útero/química , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/virologia , Linhagem Celular Tumoral , Feminino , Células HeLa , Humanos , Programas de Rastreamento/métodos , Análise Multivariada , Sensibilidade e Especificidade , Infecções Tumorais por Vírus
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