Delphine Garnier

Oncogenic epidermal growth factor receptor up-regulates multiple elements of the tissue factor signaling pathway in human glioma cells

Cancer cells frequently overexpress tissue factor (TF) and become procoagulant. This conversion may be driven by genetic transformation, including through the expression of the oncogenic epidermal growth factor receptor (EGFR) and its mutant, EGFRvIII, present in glioblastoma multiforme (GBM). Here we show that the EGFRvIII-dependent GBM cell transformation is associated with the onset of the simultaneous overexpression of TF, protease-activated receptors 1 and 2 (PAR1 and PAR2), and ectopic synthesis of factor VII (FVII). Efficient generation of factor Xa by these cells still requires exogenous FVIIa. However, as a result of EGFRvIII-dependent transformation, GBM cells become hypersensitive to TF/PAR-mediated signaling and produce ample angiogenic factors (vascular endothelial growth factor and interleukin-8) on exposure to FVIIa and PAR1- or PAR2-activating peptides. Thus, oncogenes may cause complex changes in the ability of GBM cancer cells to interact with the coagulation system, thereby exacerbating its influence on angiogenesis and disease progression.

Cancer Cells Induced to Express Mesenchymal Phenotype Release Exosome-like Extracellular Vesicles Carrying Tissue Factor

Background: Cross-talk of oncogenic and differentiation pathways in cancer coagulopathy is poorly understood. Results: EGFR activation and blockade of E-cadherin in cancer cell lines induce mesenchymal phenotype and tissue factor (TF) shedding, as exosomes, capable of transferring procoagulant activity to endothelium. Conclusion: Mesenchymal and procoagulant phenotypes are linked in cancer. Significance: Epithelial-to-mesenchymal transition (EMT) may influence tumor-vascular interactions via TF-containing exosomes. Aggressive epithelial cancer cells frequently adopt mesenchymal characteristics and exhibit aberrant interactions with their surroundings, including the vasculature. Whether the release/uptake of extracellular vesicles (EVs) plays a role during these processes has not been studied. EVs are heterogeneous membrane structures that originate either at the surface (microparticles), or within (exosomes) activated or transformed cells, and are involved in intercellular trafficking of bioactive molecules. Here, we show that epithelial cancer cells (A431, DLD-1) adopt mesenchymal features (epithelial-to-mesenchymal transition-like state) upon activation of epidermal growth factor receptor (EGFR) coupled with blockade of E-cadherin. This treatment leads to a coordinated loss of EGFR and tissue factor (TF) from the plasma membrane and coincides with a surge in emission of small, exosome-like EVs containing both receptors. TF (but not EGFR) is selectively up-regulated in EVs produced by mesenchymal-like cancer cells and can be transferred to cultured endothelial cells rendering them highly procoagulant. We postulate that epithelial-to-mesenchymal transition-like changes may alter cancer cell interactions with the vascular systems through altered vesiculation and TF shedding.

Oncogenic extracellular vesicles in brain tumor progression

The brain is a frequent site of neoplastic growth, including both primary and metastatic tumors. The clinical intractability of many brain tumors and their distinct biology are implicitly linked to the unique microenvironment of the central nervous system (CNS) and cellular interactions within. Among the most intriguing forms of cellular interactions is that mediated by membrane-derived extracellular vesicles (EVs). Their biogenesis (vesiculation) and uptake by recipient cells serves as a unique mechanism of intercellular trafficking of complex biological messages including the exchange of molecules that cannot be released through classical secretory pathways, or that are prone to extracellular degradation. Tumor cells produce EVs containing molecular effectors of several cancer-related processes such as growth, invasion, drug resistance, angiogenesis, and coagulopathy. Notably, tumor-derived EVs (oncosomes) also contain oncogenic proteins, transcripts, DNA, and microRNA (miR). Uptake of this material may change properties of the recipient cells and impact the tumor microenvironment. Examples of transformation-related molecules found in the cargo of tumor-derived EVs include the oncogenic epidermal growth factor receptor (EGFRvIII), tumor suppressors (PTEN), and oncomirs (miR-520g). It is postulated that EVs circulating in blood or cerebrospinal fluid (CSF) of brain tumor patients may be used to decipher molecular features (mutations) of the underlying malignancy, reflect responses to therapy, or molecular subtypes of primary brain tumors [e.g., glioma or medulloblastoma (MB)]. It is possible that metastases to the brain may also emit EVs with clinically relevant oncogenic signatures. Thus, EVs emerge as a novel and functionally important vehicle of intercellular communication that can mediate multiple biological effects. In addition, they provide a unique platform to develop molecular biomarkers in brain malignancies.

Extracellular vesicles in the biology of brain tumour stem cells – Implications for inter-cellular communication, therapy and biomarker development

Extracellular vesicles (EVs) act as carriers of molecular and oncogenic signatures present in subsets of tumour cells and tumour-associated stroma, and as mediators of intercellular communication. These processes likely involve cancer stem cells (CSCs). EVs represent a unique pathway of cellular export and cell-to-cell transfer of insoluble molecular regulators such as membrane receptors, signalling proteins and metabolites, thereby influencing the functional integration of cancer cell populations. While mechanisms that control biogenesis, cargo and uptake of different classes of EVs (exosomes, microvesicles, ectosomes, large oncosomes) are poorly understood, they likely remain under the influence of stress-responses, microenvironment and oncogenic processes that define the biology and heterogeneity of human cancers. In glioblastoma (GBM), recent molecular profiling approaches distinguished several disease subtypes driven by distinct molecular, epigenetic and mutational mechanisms, leading to formation of proneural, neural, classical and mesenchymal tumours. Moreover, molecularly distinct clonal cellular lineages co-exist within individual GBM lesions, where they differentiate according to distinct stem cell hierarchies resulting in several facets of tumour heterogeneity and the related potential for intercellular interactions. Glioma stem cells (GSCs) may carry signatures of either proneural or mesenchymal GBM subtypes and differ in several biological characteristics that are, at least in part, represented by the output and repertoire of EV production (vesiculome). We report that vesiculomes differ between known GBM subtypes. EVs may also reflect and influence the equilibrium of the stem cell hierarchy, contain oncogenic drivers and modulate the microenvironment (vascular niche). The GBM/GSC subtype-specific differentials in EV cargo of proteins, transcripts, microRNA and DNA may enable detection of the dynamics of the stem cell compartment and result in biological effects that remain to be fully characterized.

Tissue factor expression provokes escape from tumor dormancy and leads to genomic alterations

Significance Our study shows that the clotting protein tissue factor (TF) controls the state of tumor dormancy and does so in conjunction with recruitment of inflammatory cells and blood vessels. We show that indolent glioma cells remain harmless in mice unless rendered TF positive. Our work also demonstrates the ability of TF to indirectly influence the DNA of cancer cells by facilitating gene mutations and silencing. This ability is important because injury, cardiovascular disease, or other conditions may activate the clotting system and contribute to the awakening of occult cancer cells. This understanding also may suggest a prophylactic use of blood thinners in cases where dormant cancer cells and clotting are suspected to coexist (e.g., after surgery). The coagulation system links immediate (hemostatic) and late (inflammatory, angiogenic) tissue responses to injury, a continuum that often is subverted in cancer. Here we provide evidence that tumor dormancy is influenced by tissue factor (TF), the cancer cell-associated initiator of the coagulation system and a signaling receptor. Thus, indolent human glioma cells deficient for TF remain viable but permanently dormant at the injection site for nearly a year, whereas the expression of TF leads to a step-wise transition to latent and overt tumor growth phases, a process that is preceded by recruitment of vascular (CD105+) and myeloid (CD11b+ and F4/80+) cells. Importantly, the microenvironment orchestrated by TF expression drives permanent changes in the phenotype, gene-expression profile, DNA copy number, and DNA methylation state of the tumor cells that escape from dormancy. We postulate that procoagulant events in the tissue microenvironment (niche) may affect the fate of occult tumor cells, including their biological and genetic progression to initiate a full-blown malignancy.

Qualitative changes in the proteome of extracellular vesicles accompanying cancer cell transition to mesenchymal state

Transitions of the cancer cell phenotype between epithelial and mesenchymal states (EMT) are likely to alter the patterns of intercellular communication. In this regard we have previously documented that EMT-like changes trigger quantitative rearrangements in exosomal vesicle emission in A431 cancer cells driven by oncogenic epidermal growth factor receptor (EGFR). Here we report that extracellular vesicles (EVs) produced by these cancer cells in their epithelial and mesenchymal states exhibit profound qualitative differences in their proteome. Thus, induction of the EMT-like state through blockade of E-cadherin and EGFR stimulation provoked a mesenchymal shift in cellular morphology and enrichment in the CD44-high/CD24-low immunophenotype, often linked to cellular stemness. This change also resulted in reprogramming of the EV-related proteome (distinct from that of corresponding cells), which contained 30 unique protein signals, and revealed enrichment in pathways related to cellular growth, cell-to-cell signaling, and cell movement. Some of the most prominent EV-related proteins were validated, including integrin α2 and tetraspanin CD9. We propose that changes in cellular differentiation status translate into unique qualitative rearrangements in the cargo of EVs, a process that may have implications for intercellular communication and could serve as source of new biomarkers to detect EMT-like processes in cancer.

Role of the tissue factor pathway in the biology of tumor initiating cells.

Oncogenic transformation and aberrant cellular differentiation are regarded as key processes leading to malignancy. They produce heterogenous cellular populations including subsets of tumour initiating cells (TICs), also known as cancer stem cells (CSCs). Intracellular events involved in these changes profoundly impact the extracellular and systemic constituents of cancer progression, including those dependent on the vascular system. This includes angiogenesis, vasculogenesis, activation of the coagulation system and formation of CSC-related and premetastatic niches. Tissue factor (TF) is a unique cell-associated receptor for coagulation factor VIIa, initiator of blood coagulation, and mediator of cellular signalling, all of which influence vascular homeostasis. Our studies established a link between oncogenic events, angiogenesis and the elevated expression of TF in several types of cancer cells. The latter suggests that cancer coagulopathy and cellular events attributed to the coagulation system may have cancer-specific and genetic causes. Indeed, in human glioma cells, a transforming mutant of the epidermal growth factor receptor (EGFRvIII) triggers not only the expression of TF, but also of its ligand (factor VII) and protease activated receptors (PAR-1 and PAR-2). Consequently, tumour cells expressing EGFRvIII become hypersensitive to contact with blood borne proteases (VIIa, thrombin), which upregulate their production of angiogenic factors (VEGF and IL-8), and contribute to formation of the growth promoting microenvironment (niche). Moreover, TF overexpression accompanies features of cellular aggressiveness such as markers of CSCs (CD133), epithelial-to-mesenchymal transition (EMT) and expression of the angiogenic and prometastatic phenotype. Conversely, TF blocking antibodies inhibit tumour growth, angiogenesis, and especially tumour initiation upon injection of threshold numbers of tumourigenic cells. Likewise, TF depletion in the host compartment (e.g. in low-TF mice) perturbs tumour initiation. These observations suggest that both cancer cells and their adjacent host stroma contribute TF activity to the tumour microenvironment. We postulate that the TF pathway may play an important role in formation of the vascular niche for tumour initiating CSCs, through its procoagulant and signalling effects. Therapeutic blockade of these mechanisms could hamper tumour initiation processes, which are dependent on CSCs and participate in tumour onset, recurrence, drug resistance and metastasis.

Inhibition of oncogenic epidermal growth factor receptor kinase triggers release of exosome-like extracellular vesicles and impacts their phosphoprotein and DNA content.

Background: Cargo of extracellular vesicles (EVs) may reveal responses to targeted anticancer drugs. Results: Kinase inhibitors of the oncogenic epidermal growth factor receptor (EGFR) activate emission of exosome-like EVs containing EGFR protein and DNA. Conclusion: Co-detection of EGFR and DNA in tumor-related EVs reflects the responses to kinase inhibitors. Significance: EV cargo may serve as a biomarker for targeted therapy. Cancer cells emit extracellular vesicles (EVs) containing unique molecular signatures. Here, we report that the oncogenic EGF receptor (EGFR) and its inhibitors reprogram phosphoproteomes and cargo of tumor cell-derived EVs. Thus, phosphorylated EGFR (P-EGFR) and several other receptor tyrosine kinases can be detected in EVs purified from plasma of tumor-bearing mice and from conditioned media of cultured cancer cells. Treatment of EGFR-driven tumor cells with second generation EGFR kinase inhibitors (EKIs), including CI-1033 and PF-00299804 but not with anti-EGFR antibody (Cetuximab) or etoposide, triggers a burst in emission of exosome-like EVs containing EGFR, P-EGFR, and genomic DNA (exo-gDNA). The EV release can be attenuated by treatment with inhibitors of exosome biogenesis (GW4869) and caspase pathways (ZVAD). The content of P-EGFR isoforms (Tyr-845, Tyr-1068, and Tyr-1173), ERK, and AKT varies between cells and their corresponding EVs and as a function of EKI treatment. Immunocapture experiments reveal the presence of EGFR and exo-gDNA within the same EV population following EKI treatment. These findings suggest that targeted agents may induce cancer cells to change the EV emission profiles reflective of drug-related therapeutic stress. We suggest that EV-based assays may serve as companion diagnostics for targeted anticancer agents.

Genetic pathways linking hemostasis and cancer

Oncogenic events impact interactions of cancer cells with their surroundings. Amongst the most consequential, in this regard, is the influence on angiogenesis, inflammation and hemostasis. Indeed, mutant oncogenes (EGFR, HER2, RAS, MET, PML-RARα) are known to alter the expression of angiogenic and pro-inflammatory factors, as well as change the cancer cell coagulome, including the levels of tissue factor (TF) and other mediators (PAI-1, COX2). Accompanying losses of tumour suppressor genes (PTEN, p53), and changes in microRNA (miR-19b, miR-520) facilitate these effects. Transforming genes may also trigger ectopic production of coagulation factors (e.g. FVII) by cancer cells and their release and properties of procoagulant microparticles (MPs). By deregulating protease activated receptors (PAR1/2) oncogenes may also change tumour cell responses to coagulation factor signalling. These changes act in concert with microenvironmental factors (hypoxia), stress responses (therapy) and differentiation programs, including epithelial-to-mesechymal transitions (EMT) and through tumour initiating cell (TIC) compartment. In so doing, the coagulation system influences early (initiation, angiogenesis), intermediate (growth, invasion) and late stages (metastasis, relapse) of cancer progression. In fact, TF may act as a molecular switch that controls the transition between dormant, latent and progressive/metastatic disease. TIC-like cells may play a role in these effects, as they express TF and PAR-1/2, and respond to stimulation with their agonists. As major human malignancies (e.g. glioblastoma) are increasingly recognized to consist of a spectrum of molecularly distinct disease subtypes driven by specific genetic pathways, so too may their patterns of interaction differ with the coagulation system. A better understanding of these linkages may be a source of new diagnostic, prognostic and therapeutic opportunities.

Extracellular vesicles as prospective carriers of oncogenic protein signatures in adult and paediatric brain tumours

Extracellular vesicles (EVs), including exosomes, act as biological effectors and as carriers of oncogenic signatures in human cancer. The molecular composition and accessibility of EVs in biofluids open unprecedented diagnostic opportunities in malignancies where tumour tissue is difficult to sample, especially in primary and metastatic brain tumours. The ongoing genetic discovery of driver mutations defines the ever increasing numbers of distinct molecular subtypes of brain tumours (orphan diseases), a complexity that may soon be translated into alterations in functional proteins and their oncogenic networks. This may likely be extended to real time changes engendered by the disease progression, tumour heterogeneity, inter‐individual variations and therapeutic responses. Meeting these challenges through EV analysis is dependent on technological progress in such areas as generation of mutation‐ and phospho‐specific antibodies, antibody array platforms, nanotechnology, microfluidics, NMR spectroscopy, MS and MRM approaches of quantitative proteomics, which should not be underestimated. Still, vesiculation emerges as a unique process that could be harnessed for the benefit of more individualised patient care.