27 research outputs found
The Role of Cytokines in Support of Malignant Properties of Glioblastomas
The tumor microenvironment of Glioblastomas (GB) is very complex and transforms throughout radiotherapy and chemotherapy, posing a favourable environment for the malignant properties of GB including invasivity, which promotes aggressive recurrency of the tumor. These changes in the tumor microenvironment are partly induced by cellular senescence and cellular response to stress, accompanied by a specific secretome, so called senescence-associated secretory phenotype (SASP). The SASP is distinct in its secretion of growth factors, components of the extracellular matrix and remodelling enzymes, but above all proinflammatory cytokines, which may contribute to the malignant properties of GB. This thesis reviews the current state of knowledge of the potential role of selected cytokines of the SASP (IL-6, IL-1β, IL-8, IL-1α, TGF-β, CCL2, TNF-α) in support of the malignant properties of GB. Key words: glioblastoma (GB), senescence, SASP, malignant properties, cytokinesNádorovĂ© mikroprostĹ™edĂ glioblastomu (GB) je velice komplexnĂ a dále se mÄ›nĂ pĹ™i radioterapii a chemoterapii zpĹŻsobem, kterĂ˝ podporuje malignĂ vlastnosti glioblastomu vÄŤetnÄ› invazivity, která podporuje agresivnĂ recidivu nádoru. Na tÄ›chto zmÄ›nách nádorovĂ©ho mikroprostĹ™edĂ se podĂlĂ fenomĂ©n buněčnĂ© senescence a buněčnĂ© odpovÄ›di na stres, která je doprovázena specifickĂ˝m sekretomem, takzvanĂ˝m se senescencĂ asociovanĂ˝m sekreÄŤnĂm fenotypem (SASP). Pro SASP je typická sekrece rĹŻstovĂ˝ch faktorĹŻ, komponent extracelulárnĂ matrix a remodelujĂcĂch enzymĹŻ, ale pĹ™edevšĂm prozánÄ›tlivĂ˝ch cytokinĹŻ, kterĂ© mohou podporovat malignĂ vlastnosti GB. Tato práce je literárnĂ rešeršà úlohy vybranĂ˝ch cytokinĹŻ SASP (IL-6, IL 1β, IL-8, IL-1α, TGF-β, CCL2, TNF-α) v podpoĹ™e malignĂch vlastnostĂ glioblastomu. KlĂÄŤová slova: glioblastom (GB), senescence, SASP, malignĂ vlastnosti, cytokinyKatedra fyziologieDepartment of PhysiologyFaculty of SciencePĹ™ĂrodovÄ›decká fakult
Gene therapy strategies to enhance anti-angiogenic therapy, chemotherapy and radiotherapy in the treatment of malignant glioma a pre-clinical evaluation
Cellular and Molecular Signature of Oral Squamous Cell Carcinoma
PhDOral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. It is a result of numerous aetiological factors such as genetic predisposition, smoking, excessive alcohol consumption and viruses such as the human papilloma virus. Due to late diagnosis it has a high mortality and morbidity rates which has remained unchanged over the last 5 decades.
Currently no screening is available for high risk patients for better monitoring. Diagnosing OSCC relies on histopathology of biopsy tissue, reviewed for dysplasia and advancing lesions. Although the technique has been used for decades for successful diagnosis it fails to identify the molecular signature of OSCC which appears much before the visual signs. It also falls short in predicting the malignant transformation of pre-malignant oral lesions. Identifying the molecular and genetic changes leading to OSCC lesion will aid in more specific (quantitative) and early diagnosis of the disease reducing the financial burden of treating late-stage OSCC patients on the healthcare system.
This study focuses on developing new adjuncts which can be used alongside histopathology for early diagnosis. There is a need to monitor high risk patients through non-invasive methods causing less patient discomfort. We therefore explored the potentials of exosomes which are extracellular vesicles secreted by normal and tumour cells. They can be isolated from body fluids such as blood and saliva.
In cancer biology exosomes offer both diagnostic and therapeutic advantage. Their involvement in cell-cell communication indicates their influence in tumour development, progression, metastasis and therapeutic efficacy. Exosomes released by cancerous cells carry numerous biomarkers, which are passed on to healthy cells via microenvironment, causing stromal and angiogenic activation along with immune escape.
In this study exosomes were successfully isolated from body fluids (blood, saliva and plasma) and cell line supernatant through ultracentrifugation and characterised by visual and particle size quantification techniques including Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM), Zetasizer and Nanosight Tracking Analysis (NTA). Exosomal specific membrane proteins were identified through Western blotting.
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We report the presence of a potential protein biomarker located exclusively on the outer membrane of cancer exosomes. Since body fluids consist of a heterogeneous population of exosomes derived from multiple cell types, such surface biomarker can potentially be used to isolate OSCC exosomes.
Characterisation of exosomal mRNA cargo was done using Agilent Bioanalyzer (for RNA quantity and quality assurance) and reverse transcription-quantitative PCR (RT-qPCR; for gene specific quantitation). Functional significance of exosomes was studied by transfecting normal oral keratinocyte cells with self and cancer-derived exosomes. Through gene-expression microarray and subsequent RT-qPCR verification, we report a panel of differentially expressed genes involved in essential cellular functions being modulated by exosome transfection.
A previously developed molecular diagnostic system by our research group called quantitative malignancy index diagnostic system (qMIDS) based on FOXM1 oncogene and its downstream targets was validated on archival formalin fixed paraffin embedded OSCC patient biopsy samples. We report that qMIDS index successfully correlates with the disease stages including dysplasia, tumour and lymph node metastasis.
Furthermore, through meta-analysis of 8 OSCC microarray studies we identified a panel of six genes including PLAU, FN1, CDCA5, CRNN, CLEC3B and DUOX1 (q6) which are able to identify two clinically distinct sub-groups of OSCC patient population. Through RT-qPCR the expression of q6 biomarkers was established in 100 OSCC biopsy samples. This information can be of immense importance in developing personalized treatment strategies based on the molecular makeup of the presenting tumour
LIV-1 Promotes Prostate Cancer Epithelial-to-Mesenchymal Transition and Metastasis Through HB-EGF Shedding and EGFR-mediated ERK Signaling
LIV-1, a zinc transporter, is an effector molecule downstream from soluble growth factors. This protein has been shown to promote epithelial-to-mesenchymal transition (EMT) in human pancreatic, breast, and prostate cancer cells. Despite the implication of LIV-1 in cancer growth and metastasis, there has been no study to determine the role of LIV-1 in prostate cancer progression. Moreover, there is no clear delineation of the molecular mechanism underlying LIV-1 function in cancer cells. In this study, we found increased LIV-1 expression in a progresssive manner in benign, PIN, primary and bone metastatic human prostate cancer. We characterized the mechanism by which LIV-1 drives prostate cancer EMT in an androgen-refractory human prostate cancer cell (ARCaP) bone metastasis model. LIV-1, when overexpressed in ARCaPE cells (derivative cells of ARCaP with epithelial phenotype), promoted EMT irreversibly. LIV-1 overexpressed ARCaPE cells had elevated levels of HB-EGF and matrix metalloproteinase (MMP) 2 and MMP 9 proteolytic enzyme activities, without affecting intracellular zinc concentration. The activation of MMPs resulted in the shedding of heparin binding-epidermal growth factor (HB-EGF) from ARCaPE cells, eliciting constitutive epidermal growth factor receptor (EGFR) phosphorylation and its downstream extracellular signal regulated kinase (ERK) signaling. Further investigation of the HB-EGF promoter revealed that both Stat3 and AP-1 controlled HB-EGF promoter activity. Ectopic LIV-1 overexpression induced AP-1 and Stat3 activation. Blockade of both Stat3 and AP-1 by specific inhibitors or dominant negative expression vectors diminished the HB-EGF promoter activity induced by LIV-1 overexpression. These results suggest that LIV-1 is involved in prostate cancer progression as an intracellular target of growth factor receptor signaling which promotes EMT and cancer metastasis. LIV-1 could be an attractive therapeutic target for the eradication of pre-existing human prostate cancer and bone and soft tissue metastases
QUANTITATIVE STUDY ON REGULATORY MECHANISMS OF CELL PHENOTYPE TRANSITION
Cells in a multicellular organism share the same set of genome and can assume multiple phenotypes. Uncovering mechanisms of regulating cell phenotype changes has become an important and active research area. This dissertation presents a collection of my combined computational and experimental efforts on studying cell phenotype transitions.
Chapter I gives a literature overview on cell phenotype conversion and regulation. One can identify four generic modules that function coordinately to regulate cell phenotypes. The whole system forms a highly interconnected network and involves a large number of molecular species for epigenetic, transcriptional and translational regulations.
Chapter II addresses how a cell interprets temporal and strength information of signals and makes cell fate decision. I performed an integrated quantitative and computational analysis on how extracellular TGF-β signal is transmitted intracellularly to activate SNAIL1 expression. I demonstrated how quantitative information of TGF-β is distributed through upstream divergent pathways then crosstalk at various places and converge on to SNAIL1. This crosstalk network interprets the duration of TGF-β signal and is robust against stochastic fluctuations.
Chapter III and IV focus on co-regulation of multiple genes that orchestrate cell functions and phenotype changes. In eukaryotic cells, the expression level of a gene is determined by both transcription factors and the local environment, such as histone modifications and three-dimensional chromosome structure. I used EMT in human cell line and neural cell differentiation process in mouse as model systems, respectively. Through performing combined analysis of data of gene expression, epigenetic modification and chromosome conformation, I examined how the local environment and transcriptional factor regulation is coupled. I discovered that genes co-regulated by a common transcription factor (TF) has tendency to be close both sequentially and spatially. The local spatial organization bridged by TFs is cell type specific. Reorganization of DNA local conformation has impact on gene co-regulation during cell phenotype transition.
The final chapter gives a brief summary of the conclusion from my computational and experimental researches in this dissertation. In this chapter, I also introduced the future work in investigating the relationship between chromosome conformation and gene regulation during cell type transition
AXL receptor tyrosine kinase in breast cancer : defining novel substrates and pathways involved in cell motility and invasion
Le cancer du sein est le cancer le plus fréquemment diagnostiqué et le plus mortelle chez la femme, où sa progression vers le stade métastatique constitue une menace pour la vie des patientes. La présence de métastases représente le défi clinique central de l'oncologie des tumeurs solides, de sorte que les mécanismes et les voies sous-jacents au processus métastatique doivent être mieux définis. L'expression aberrante du récepteur tyrosine kinase (RTK) AXL a été liée cliniquement à la formation de métastases et à l'acquisition d'une résistance aux médicaments contre le cancer. AXL est un membre de la sous-famille des récepteurs tyrosine kinase TAM et intervient dans plusieurs processus biologiques tels que l'atténuation de la réponse immunitaire, l'élimination des cellules apoptotiques et la promotion de la survie cellulaire. L'expression d'AXL dans les tumeurs primaires humaines corrèle avec la faible survie des patients. Malgré sa régulation positive préférentielle dans les lignées cellulaires triple négatives / basales B, des études ont montré que l’expression d’AXL est indépendante du sous-type de la tumeur mammaire des patients. AXL peut être activé par son ligand GAS6 ou par d'autres RTK. Lors de son activation, AXL induit une signalisation en aval entraînant l'activation d'intermédiaires de signalisation canoniques, notamment MAPK, AKT et PI 3-kinases. Cependant, les voies de signalisation spécifiques engagées par AXL pour conférer un tel pouvoir pro-invasion ne sont pas connues. Ainsi, le but de cette thèse est d'identifier des substrats spécifiques d’AXL et des voies en aval qui jouent un rôle important dans le maintien d'un état « EMT » et d'un renforcement du phénotype mésenchymal dans les cellules cancéreuses.
À la recherche de régulateurs en amont du complexe ELMO/DOCK1 impliqués dans l’activation de RAC, nous présentons au chapitre 2 les protéines d’échafaudage ELMO en tant que substrats directs et partenaires de liaison d’AXL. Grâce à des approches de protéomique et de mutagenèse, nous révélons que la kinase AXL phosphoryle ELMO1/2 sur un résidu tyrosine carboxy-terminal conservé. Dans les cellules cancéreuses du sein, l'activation d'AXL dépendante de GAS6 a conduit à la phosphorylation endogène d'ELMO2 sur Tyr-713, menant ainsi à la formation du complexe AXL/ELMO. En outre, l'activation de RAC induite par GAS6 dans les cellules cancéreuses du sein dépendait de l'expression d'ELMO2. Semblable au blocage d’AXL, l'inhibition d’ELMO2 ou l'inhibition pharmacologique de DOCK1 supprime l'invasion des cellules du cancer du sein, qui, selon nous, dépendait de l'état de phosphorylation d'ELMO. Notre travail au chapitre 2 définit un nouveau mécanisme par lequel AXL favorise la prolifération et l'invasion cellulaire et identifie l'inhibition de la voie ELMO/DOCK comme une cible thérapeutique potentielle pour arrêter les métastases induites par AXL.
Bien qu'il soit encore difficile de savoir comment les signaux d’AXL induisent son phénotype pro-invasif, notre travail au Chapitre 3 vise à identifier des substrats et des voies de signalisation spécifiques qui sont significativement modulés lors de l'activation d'AXL. Pour y remédier, nous avons défini le phosphoprotéome de la régulation d’AXL dans des cellules cancéreuses du sein triple-négatives en utilisant une approche quantitative. Nous révélons qu’AXL module de manière robuste, parmi de nombreux processus et voies biologiques importants, la phosphorylation d'un réseau de protéines d'adhésion focale (FA) aboutissant à un désassemblage plus rapide des FA. De manière intéressante, nous avons trouvé que la modulation de la voie FA était unique à AXL par rapport à d'autres RTK tels que l'EGFR. En particulier, nous avons trouvé qu’AXL phosphoryle la protéine NEDD9, modulant la formation du complexe NEDD9/CRKII/DOCK3, qui orchestre la phosphorylation de la pseudo-kinase PEAK1 médiée par AXL. Nos données révèlent un mécanisme distinct par lequel les complexes PEAK1 avec la kinase CSK médient la phosphorylation de PXN et le renouvellement des FA induit par AXL. En utilisant l'injection orthotopique de cellules cancéreuses du sein dans le tissu adipeux mammaire des souris et dans la veine de la queue, nous révélons que l'inactivation de PEAK1 par CRISPR diminue la croissance tumorale et les métastases in vivo. De plus, notre travail au chapitre 3 révèle une contribution unique et inattendue de la signalisation d’AXL à la dynamique des FA, révélant un mécanisme longtemps recherché sous-tendant l'activité invasive d'AXL. Cette compréhension approfondie des réseaux de signalisation régulés par AXL identifie PEAK1 comme une nouvelle cible thérapeutique dans les tumeurs AXL positives.
En conclusion, cette thèse a identifié, pour la première fois, le phosphoprotéome d’AXL et des voies de signalisation spécifique à AXL, pouvant justifier le rôle du récepteur en tant que promoteur de métastases et de résistance aux médicaments. Notre travail révèle de nouvelles cibles thérapeutiques qui pourraient avoir un grand potentiel si elles sont utilisées en thérapie combinatoire avec l’inhibition d’AXL pour prévenir la formation de métastases des tumeurs AXL positives.Breast cancer is the most frequently diagnosed cancer in women where its progression to the metastatic stage poses a threat to the life of patients. The metastatic disease represents the central clinical challenge of solid tumor oncology such that mechanisms and pathways underlying the metastatic process must be better defined. The aberrant expression of the receptor tyrosine kinase (RTK) AXL has been linked clinically to metastasis and acquisition of drug resistance. AXL is a member of the TAM subfamily and functions in several biological processes such as dampening the immune response, clearing apoptotic cells and promoting cell survival. Despite its preferential upregulation in triple negative/basal B cell lines, studies have shown AXL expression in the clinic to be subtype independent. AXL can be activated by its ligand GAS6 or by a crosstalk with other RTKs. Upon its activation, AXL induces downstream signaling resulting in the activation of canonical signaling intermediates including MAPKs, AKT and PI 3-kinases. However, the specific signaling pathways engaged by AXL to confer such enhanced pro-invasion power are not known and the goal of this thesis is to identify AXL-specific substrates and downstream pathways that are behind AXL’s significant role in maintaining an EMT state and reinforced mesenchymal phenotype in cancer cells.
In search of upstream regulators of ELMO/DOCK1 complex involved in RAC activation, we reported ELMO scaffolds as direct substrates and binding partners of AXL. Through proteomics and mutagenesis approaches, we revealed phosphorylation of ELMO1/2 by AXL kinase on a conserved carboxyl-terminal tyrosine residue. In breast cancer cells, GAS6-dependent activation of AXL led to endogenous ELMO2 phosphorylation on Tyr-713 and AXL/ELMO complex formation. In addition, GAS6-induced RAC activation in breast cancer cells was dependent on ELMO2 expression and phosphorylation. Our work in chapter 2 defines a new mechanism by which AXL promotes cell proliferation and invasion and identifies inhibition of ELMO/DOCK pathway as a potential therapeutic target to stop AXL-induced metastases.
While it still remains elusive how AXL signals to induce its pro-invasive phenotype, our work strove to identify specific substrates and signaling pathways that are significantly modulated upon AXL activation using a quantitative phosphoproteomics approach. By generating GAS6-induced AXL phosphoproteome, we found that AXL robustly modulates, among many different significant biological processes and pathways, the phosphorylation of a network of focal adhesion (FA) proteins culminating in faster FA disassembly. Interestingly, we found AXL modulation of FA pathway to be unique to AXL in comparison with other RTKs such as EGFR. NEDD9 FA protein was identified to be a direct substrate of AXL, where its phosphorylation modulates its complex formation with CRKII/DOCK3, and this subsequently orchestrates the AXL-mediated phosphorylation of the pseudo-kinase PEAK1. Our data revealed a distinct mechanism by which PEAK1 complexes with CSK kinase, mediating PXN phosphorylation and AXL-induced FA turnover. Using in vivo assays such as tail-vein metastasis assay and tumor growth assay, we revealed that gene inactivation of PEAK1 by CRISPR CAS9 decreased tumor growth and metastasis. Furthermore, our work in chapter 3 uncovers an unexpected and unique robust contribution of AXL signaling to FA dynamics revealing a long sought-after mechanism underlying AXL pro-invasive activity. This in-depth understanding of AXL regulated signaling networks identifies PEAK1 as a new therapeutic target in AXL positive tumors.
In conclusion, this thesis identified, for the first time, AXL phosphoproteome and AXL specific downstream signaling pathways that may justify AXL’s role as a promoter of metastasis and drug resistance. Our work reveals novel therapeutic drug targets that may hold a great potential if used in combinational therapeutics with AXL inhibition to prevent metastasis of AXL positive tumors
THE ROLE OF TUMOR DESMOPLASIA IN NANOPARTICLE DELIVERY OF DRUGS AND GENES
In desmoplastic tumors, stroma cells capture nanoparticles (NPs), preventing them from reaching tumor cells, resulting in compromised anti-tumor efficacy. This dissertation focuses on understanding the basis role of tumor associated fibroblasts (TAFs), one of the major stroma cells constituting desmoplasia, in NP delivery and tumor resistance, as well as proposing strategies to overcome the TAF-elicited barriers and improve efficacy. While the capture of therapeutic NPs in TAFs interferes tumor-stroma crosstalk and inhibits tumor progression, we found that the chronic exposure of NPs paradoxically induced the secretion of survival factors (e.g., Wnt16) from the damaged TAFs, facilitating tumor proliferation and metastasis. Therefore, we proposed the delivery of siRNA against Wnt16 to TAFs via the off-target capture, to downregulate this survival factor. The priming of damaged fibroblasts could synergize with a nanoformulation of cisplatin, and benefit the treatment of a desmoplastic bladder cancer xenograft (UMUC3/3T3). Since the off-target delivery of NPs have been verified, we further utilized the same rationale to generate a group of tumor-suppressive TAFs through transfecting TAFs with a plasmid encoding highly secretable TNF-related apoptosis-inducing ligand (sTRAIL). The production of sTRAIL from TAFs bypassed the stroma barrier and resulted in efficient killing of tumor cells. Furthermore, we also proposed a stroma depletion method via combination therapy of cisplatin NPs and gemcitabine NPs. This combination was not only detrimental to tumor cells, but induced superior apoptosis in TAFs of the UMUC3/3T3 model. To ensure the sufficient synergy, we further designed a nano-formulation with ratiometric co-loading and co-delivery of these two regimens. The design of converting these two drugs with totally different physicochemical properties into nano-cores with similar hydrophobic surface and particle size, allows for their simultaneously and ratiometric loading in a single PLGA NPs. This combinatory NPs showed potent anti-cancer efficacy compared to each regimens in separate NPs. In summary, the stroma modulating strategies proposed in the current dissertation provide new paradigms for the treatment of desmoplastic tumors. Combined with cancer immunology, a more prolonged and efficient outcome can be anticipated. The ratiometric combination nano-platform also provides a promising approach for encapsulating agents with different physicochemical properties.Doctor of Philosoph
Role of miRNAs in Cancer
MicroRNAs are the best representatives of the non-coding part of the genome and their functions are mostly linked to their target genes. During the process of carcinogenesis, both dysregulation of microRNAs and their target genes can explain the development of the disease. However, most of the target genes of microRNAs have not yet been elucidated. In this book, we add new information related to the functions of microRNAs in various tumors and their associated targetome
The HBP1 tumor suppressor is a negative epigenetic regulator of MYCN driven neuroblastoma through interaction with the PRC2 complex
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