Characterization of proteins, mRNAs, and miRNAs of circulating extracellular vesicles from prostate cancer patients compared to healthy subjects.

Prostate cancer (PC) is the fifth leading cause of death in men globally. Measurement of the blood PSA level is still considered the gold-standard biomarker test for PC despite its high rate of delivering false positives and negatives that result in an inappropriate medical response, including overtreatment. We collected extracellular vesicles (EVs) from the blood plasma of PC patients with organ-confined, extracapsular-invading, and seminal vesicle-invading tumors and from healthy subjects. We examined the protein, mRNA, and miRNA content of these EVs using mass spectrometry (MS), a human PC PCR array, and a miScript miRNA PCR array, respectively. The proteomic analysis showed distinct groups of proteins that are differently expressed in each group of patients, as well as in healthy subjects. Samples from healthy subjects and each tumor type were used for both mRNA and miRNA arrays. The mRNA analysis showed distinct groups of mRNAs that were overexpressed in healthy or in one of the three tumor types but not in the EVs of the other groups. The miRNA analysis showed distinct groups of miRNAs as well. The fold of regulation in the expression of the identified mRNA and miRNA of each stage of the disease from healthy subjects showed that various mRNAs and miRNAs could discriminate the disease stage. Overall, our data suggest many molecular marker candidates for distinguishing between healthy subjects and PC patients using the cargo of circulating vesicles, as well as markers to discriminate between the different tumor types. Once verified, these markers might have a diagnostic value for PC.

Circulating microvesicle protein is associated with renal transplant outcome.

Renal transplantation is an effective therapy with improved long-term outcomes compared with other therapies for end stage renal disease. Present methods for evaluating kidney allograft function, such as serum creatinine or allograft biopsy, are not sensitive and identify pathological changes only after any potential intervention would be effective. Thus, there is a necessity for biomarkers that would provide early prognostic information about kidney transplant outcomes. Circulating microvesicles represent an attractive source of biomarkers for different diseases including renal failure. We have studied the proteins of the circulating microvesicles from two populations of kidney transplant recipients (n = 20) with poor transplant outcomes (n = 10) or good transplant outcome (n = 10), according to their estimated glomerular filtration rate (eGFR). Microvesicles from age-matched healthy subjects (n = 10) were used as a control. Also, we performed a pilot study to assess the microvesicle protein in kidney transplant recipients before and six months after kidney transplant (n = 6), compared to healthy subjects. Proteomic analysis of microvesicles could discriminate between transplant recipients and healthy subjects, and between transplant patients based on eGFR. Our results shed light on the potential of blood microvesicles to provide a novel tool for the prediction of the outcome of kidney transplants.

The role of circulating extracellular vesicles in breast cancer classification and molecular subtyping.

Currently, tumor biopsies are used for breast cancer molecular subtyping. Biopsies are associated with various pathological changes and are thought to contribute to the dissemination of tumor cells. Extracellular vesicles shed by tumor cells into circulation exhibit the molecular signature of the parent cells. Herein, we show that proteomic analysis of circulating EV can discriminate BC patients from healthy subjects and indicate stage of the disease. Also, we performed a correlation between the BC molecular subtype using plasma EV and immunohistochemistry of tumor biopsies. Circulating EV may represent a useful, non-invasive tool to study the molecular makeup of BC tumors.

Thrombotic characteristics of extracellular vesicles derived from prostate cancer cells.

BACKGROUND: Prostate cancer (PC) patients in advanced stages of the disease have high risk of blood coagulation complications. The procoagulant molecule Tissue factor (TF), and the fibrinolysis inhibitor plasminogen activator inhibitor-1 PAI-1 play important role in this complication. Extracellular vesicles (EV) shed from cancer cells may contribute to the regulation of TF and PAI-1. The procoagulant activity of EV can be associated with the oncogenic and metastatic characteristics of their cells. METHODS: We have expressed EGFRvIII in DU145 cells to assess the role of this oncogene in the procoagulant activity of EV. The intercellular exchange of TF via EV was assessed by downregulating its expression in DU145 cells using shRNA vector, and determining the transfer of TF via EV enriched with the protein. Two PC cell lines with different metastatic potential were used to assess the correlation between the procoagulant activity of EV and the metastatic potential of PC cells. Photometric assays were used to determine FXa-activity and thrombin generation as indicators for the procoagulant activity of EV. Double-tagged proteinase-activated receptor 1(PAR-1) expressed in CHO cells to assess its activation by EV. RESULTS: The expression of EGFRvIII in DU145 cells led to increased mRNA levels for TF and PAI-1, but the increase in these proteins expression was detected mostly in the EV. EV with enhanced levels of TF protein conferred higher TF procoagulant activity on the acceptor cells by intercellular exchange of this protein. Procoagulant activity of EV, assessed by FXa activity, and thrombin generation, was correlated with the oncogenic and metastatic potential of PC cells. The ability of EV to generate thrombin led to the activation of PAR-1, which was evident by the truncation of tagged-PAR-1. CONCLUSION: The active oncogene EGFRvIII increases the concentration of TF and PAI-1 in EV. The procoagulant activity of EV is associated with the oncogenic and metastatic characteristics of their PC cells. Also, EV may contribute to the high procoagulant activity in the tumour microenvironment by the intercellular exchange of TF. Finally, through the generation of thrombin, EV can activate PAR-1, which evidently contributes to cancer progression, linking the coagulation system to tumor progression.

Overexpression of MUC1 and Genomic Alterations in Its Network Associate with Prostate Cancer Progression.

We investigate the association of MUC1 with castration-resistant prostate cancer (CRPC), bone metastasis, and PC recurrence. MUC1 expression was studied in patient-derived bone metastasis and CRPCs produced by prostate-specific PTEN-/- mice and LNCaP xenografts. Elevations in MUC1 expression occur in CRPC. Among nine patients with hormone-naïve bone metastasis, eight express MUC1 in 61% to 100% of PC cells. Utilizing cBioPortal PC genomic data, we organized a training (n=300), testing (n=185), and validation (n=194) cohort. Using the Cox model, a nine-gene signature was derived, including eight genes from a MUC1-related network (APC, CTNNB1/ß-catenin, GALNT10, GRB2, LYN, SIGLEC1, SOS1, and ZAP70) and FAM84B. Genomic alterations in these genes reduce disease-free survival (DFS) in the training (P=.00161), testing (P=.00699), entire (training+testing, P=5.557e-5), and a validation cohort (P=3.326e-5). The signature independently predicts PC recurrence [hazard ratio (HR)=1.731; 95% confidence interval (CI): 1.104-2.712; P=.0167] after adjusting for known clinical factors and stratifies patients with high risk of PC recurrence using the median (HR 2.072; 95% CI: 1.245-3.450, P=.0051) and quartile 3 (HR 3.707, 95% CI: 1.949-7.052, P=6.51e-5) scores. Several novel ß-catenin mutants are identified in PCs leading to a rapid onset of death and recurrence. Genomic alterations in APC and CTNNB1/ß-catenin reduce DFS in two independent PC cohorts (n=485, P=.0369; n=84, P=.0437). The nine-gene signature also associates with reductions in overall survival (P=.0458) and DFS (P=.0163) in melanoma patients (n=367). MUC1 upregulation is associated with CRPC and bone metastasis. A nine-gene signature derived from a MUC1 network predicts PC recurrence.

Microvesicles Contribute to the Bystander Effect of DNA Damage.

Genotoxic treatments elicit DNA damage response (DDR) not only in cells that are directly exposed but also in cells that are not in the field of treatment (bystander cells), a phenomenon that is commonly referred to as the bystander effect (BE). However, mechanisms underlying the BE remain elusive. We report here that etoposide and ultraviolet (UV) exposure stimulate the production of microvesicles (MVs) in DU145 prostate cancer cells. MVs isolated from UV-treated DU145 and A431 epidermoid carcinoma cells as well as etoposide-treated DU145 cells induced phosphorylation of ataxia-telangiectasia mutated (ATM) at serine 1981 (indicative of ATM activation) and phosphorylation of histone H2AX at serine 139 (γH2AX) in naïve DU145 cells. Importantly, neutralization of MVs derived from UV-treated cells with annexin V significantly reduced the MV-associated BE activities. Etoposide and UV are known to induce DDR primarily through the ATM and ATM- and Rad3-related (ATR) pathways, respectively. In this regard, MV is likely a common source for the DNA damage-induced bystander effect. However, pre-treatment of DU145 naïve cells with an ATM (KU55933) inhibitor does not affect the BE elicited by MVs isolated from etoposide-treated cells, indicating that the BE is induced upstream of ATM actions. Taken together, we provide evidence supporting that MVs are a source of the DNA damage-induced bystander effect.

Sterol Regulatory Element Binding Protein (SREBP)-1 is a novel regulator of the Transforming Growth Factor (TGF)-ß receptor I (TßRI) through exosomal secretion.

Accumulation of matrix in the glomerulus is a classic hallmark of diabetic nephropathy. The profibrotic cytokine transforming growth factor beta 1 (TGF-ß1) plays a central role in the development of glomerular sclerosis. Recent studies have demonstrated that the transcription factor sterol regulatory element binding protein (SREBP)-1 is an important regulator of glomerular sclerosis through both induction of TGF-ß1 as well as facilitation of its signaling. Here we have identified that SREBP-1 is also a novel regulator of TGF-ß receptor I (TßRI) expression in kidney mesangial cells. Inhibition of SREBP activation with fatostatin or downregulation of SREBP-1 using siRNA inhibited the expression of the receptor. SREBP-1 did not regulate TßRI transcription, nor did it induce its proteasomal or lysosomal degradation or proteolytic cleavage. Disruption of lipid rafts with cyclodextrin, however, prevented TßRI downregulation. This was not dependent on caveolae since SREBP-1 inhibition could induce TßRI downregulation in caveolin-1 knockout mesangial cells. SREBP-1 associated with TßRI, and SREBP-1 inhibition led to the secretion of TßRI in exosomes. Thus, we have identified a novel role for SREBP-1 as a cell surface retention factor for TßRI in mesangial cells, preventing its secretion in exosomes. Inhibition of SREBP-1 in vivo may thus provide a novel therapeutic strategy for diabetic nephropathy which targets multiple aspects of TGFß signaling and matrix upregulation.

Nuclear transportation of exogenous epidermal growth factor receptor and androgen receptor via extracellular vesicles.

Epidermal growth factor receptor (EGFR) plays a central role in the progression of several human malignancies. Although EGFR is a membrane receptor, it undergoes nuclear translocation, where it has a distinct signalling pathway. Herein, we report a novel mechanism by which cancer cells can directly transport EGFR to the nucleus of other cells via extracellular vesicles (EVs). The transported receptor is active and stimulates the nuclear EGFR pathways. Interestingly, the translocation of EGFR via EVs occurs independently of the nuclear localisation sequence that is required for nuclear translocation of endogenous EGFR. Also, we found that the mutant receptor EGFRvIII could be transported to the nucleus of other cells via EVs. To assess the role of EVs in the regulation of an actual nuclear receptor, we studied the regulation of androgen receptor (AR). We found that full-length AR and mutant variant ARv7 are secreted in EVs derived from prostate cancer cell lines and could be transported to the nucleus of AR-null cells. The EV-derived AR was able to bind the androgen-responsive promoter region of prostate specific antigen, and recruit RNA Pol II, an indication of active transcription. The nuclear-translocated AR via EVs enhanced the proliferation of acceptor cells in the absence of androgen. Finally, we provide evidence that nuclear localisation of AR could occur in vivo via orthotopically-injected EVs in male SCID mice prostate glands. To our knowledge, this is the first study showing the nuclear translocation of nuclear receptors via EVs, which significantly extends the role of EVs as paracrine transcriptional regulators.

Analysis of Extracellular Vesicles in the Tumor Microenvironment.

Extracellular vesicles (ECV) are membrane compartments shed from all types of cells in various physiological and pathological states. In recent years, ECV have gained an increasing interest from the scientific community for their role as an intercellular communicator that plays important roles in modifying the tumor microenvironment. Multiple techniques have been established to collect ECV from conditioned media of cell culture or physiological fluids. The gold standard methodology is differential centrifugation. Although alternative techniques exist to collect ECV, these techniques have not proven suitable as a substitution for the ultracentrifugation procedure.

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

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.

Gut commensal microvesicles reproduce parent bacterial signals to host immune and enteric nervous systems.

Ingestion of a commensal bacteria, Lactobacillus rhamnosus JB-1, has potent immunoregulatory effects, and changes nerve-dependent colon migrating motor complexes (MMCs), enteric nerve function, and behavior. How these alterations occur is unknown. JB-1 microvesicles (MVs) are enriched for heat shock protein components such as chaperonin 60 heat-shock protein isolated from Escherichia coli (GroEL) and reproduce regulatory and neuronal effects in vitro and in vivo. Ingested labeled MVs were detected in murine Peyer's patch (PP) dendritic cells (DCs) within 18 h. After 3 d, PP and mesenteric lymph node DCs assumed a regulatory phenotype and increased functional regulatory CD4(+)25(+)Foxp3+ T cells. JB-1, MVs, and GroEL similarly induced phenotypic change in cocultured DCs via multiple pathways including C-type lectin receptors specific intercellular adhesion molecule-3 grabbing non-integrin-related 1 and Dectin-1, as well as TLR-2 and -9. JB-1 and MVs also decreased the amplitude of neuronally dependent MMCs in an ex vivo model of peristalsis. Gut epithelial, but not direct neuronal application of, MVs, replicated functional effects of JB-1 on in situ patch-clamped enteric neurons. GroEL and anti-TLR-2 were without effect in this system, suggesting the importance of epithelium neuron signaling and discrimination between pathways for bacteria-neuron and -immune communication. Together these results offer a mechanistic explanation of how Gram-positive commensals and probiotics may influence the host's immune and nervous systems.

Regulation of the tumor suppressor PTEN through exosomes: a diagnostic potential for prostate cancer.

PTEN is a potent tumor-suppressor protein. Aggressive and metastatic prostate cancer (PC) is associated with a reduction or loss of PTEN expression. PTEN reduction often occurs without gene mutations, and its downregulation is not fully understood. Herein, we show that PTEN is incorporated in the cargo of exosomes derived from cancer cells. PTEN is not detected in exosomes derived from normal, noncancerous cells. We found that PTEN can be transferred to other cells through exosomes. In cells that have a reduction or complete loss of PTEN expression, the transferred PTEN is competent to confer tumor-suppression activity to acceptor cells. In PC patients, we show that PTEN is incorporated in the cargo of exosomes that circulate in their blood. Interestingly, normal subjects have no PTEN expression in their blood exosomes. Further, we found that the prostate-specific antigen (PSA) is incorporated in PC patients' and normal subjects' blood exosomes. These data suggest that exosomal PTEN can compensate for PTEN loss in PTEN deficient cells, and may have diagnostic value for prostate cancer.

Microvesicles as mediators of intercellular communication in cancer--the emerging science of cellular 'debris'.

Cancer cells emit a heterogeneous mixture of vesicular, organelle-like structures (microvesicles, MVs) into their surroundings including blood and body fluids. MVs are generated via diverse biological mechanisms triggered by pathways involved in oncogenic transformation, microenvironmental stimulation, cellular activation, stress, or death. Vesiculation events occur either at the plasma membrane (ectosomes, shed vesicles) or within endosomal structures (exosomes). MVs are increasingly recognized as mediators of intercellular communication due to their capacity to merge with and transfer a repertoire of bioactive molecular content (cargo) to recipient cells. Such processes may occur both locally and systemically, contributing to the formation of microenvironmental fields and niches. The bioactive cargo of MVs may include growth factors and their receptors, proteases, adhesion molecules, signalling molecules, as well as DNA, mRNA, and microRNA (miRs) sequences. Tumour cells emit large quantities of MVs containing procoagulant, growth regulatory and oncogenic cargo (oncosomes), which can be transferred throughout the cancer cell population and to non-transformed stromal cells, endothelial cells and possibly to the inflammatory infiltrates (oncogenic field effect). These events likely impact tumour invasion, angiogenesis, metastasis, drug resistance, and cancer stem cell hierarchy. Ongoing studies explore the molecular mechanisms and mediators of MV-based intercellular communication (cancer vesiculome) with the hope of using this information as a possible source of therapeutic targets and disease biomarkers in cancer.

Tissue factor and cancer stem cells: is there a linkage?

A common feature in the progression of multiple human malignancies is the protracted deregulation of the coagulation system, often referred to as cancer coagulopathy. Indeed, cancer cells and their vascular stroma often exhibit procoagulant properties, of which deregulation of tissue factor (TF) expression is a notable, although not the sole example. These changes can be traced to oncogenic influences affecting epidermal growth factor receptor (EGFR), EGFRvIII, K-ras, p53, PTEN, and probably many other proto-oncogenes and tumor suppressors in tumor parenchyma. Cancer stem cells (CSCs)/tumor initiating cells (TICs) are thought to represent the primary target and the main cellular effector through which oncogenic mutations exert their tumor-inducing effects. In so doing, CSCs/TICs depend on interactions with the tumor vasculature, which forms supportive niches for their clonal growth. We postulate that TF contributes to these interactions (directly or indirectly) through procoagulant and signaling effects, the latter executed in concert with juxtaposed protease activated receptors (mainly PAR-1 and PAR-2). TF/PAR system acts as a "blood sensing" mechanism, whereby cancer cells, including CSCs/TICs, may respond to plasma proteases (Factors VIIa, Xa, and IIa) and their related microenvironmental changes (fibrin deposition, activation of platelets). A growing body of still largely circumstantial evidence suggests that these events may contribute to the CSC/TIC niche, which could influence tumor initiation, metastasis, recurrence, and therapeutic intractability. Indeed, certain types of cancer cells harboring markers of CSCs (CD133) exhibit elevated TF expression and depend on this receptor to efficiently initiate tumor growth. We propose that both tumor cell-associated and host-related TF could influence the properties of CSCs, and that agents targeting the TF/PAR system may represent a hitherto unappreciated therapeutic opportunity to control cancer progression by influencing the CSC/TIC compartment.

Microvesicles: messengers and mediators of tumor progression.

Cellular interactions play a crucial role in progression, angiogenesis and invasiveness of tumors, including glioma. The traditionally accepted view is that medium and long-range cellular communications occur primarily through gradients of soluble ligands, recognizable by the cell-associated receptors. Recent findings, however, suggest the existence of another mode of intercellular communication, where the 'units' of information are microvesicles containing a multitude of biologically active protein and RNA species, including oncogenic receptors, such as EGFRvIII. Moreover, microvesicles can be retrieved from the circulating blood of cancer patients, and reveal the presence of oncogenes in their tumors, thereby potentially serving as information-rich prognostic and predictive biomarkers.

Endothelial expression of autocrine VEGF upon the uptake of tumor-derived microvesicles containing oncogenic EGFR.

Activated EGF receptor (EGFR) plays an oncogenic role in several human malignancies. Although the intracellular effects of EGFR are well studied, its ability to induce and modulate tumor angiogenesis is less understood. We found previously that oncogenic EGFR can be shed from cancer cells as cargo of membrane microvesicles (MVs), which can interact with surfaces of other cells. Here we report that MVs produced by human cancer cells harboring activated EGFR (A431, A549, DLD-1) can be taken up by cultured endothelial cells, in which they elicit EGFR-dependent responses, including activation of MAPK and Akt pathways. These responses can be blocked by annexin V and its homodimer, Diannexin, both of which cloak phosphatidylserine residues on the surfaces of MVs. Interestingly, the intercellular EGFR transfer is also accompanied by the onset of VEGF expression in endothelial cells and by autocrine activation of its key signaling receptor (VEGF receptor-2). In A431 human tumor xenografts in mice, angiogenic endothelial cells stain positively for human EGFR and phospho-EGFR, while treatment with Diannexin leads to a reduction of tumor growth rate and microvascular density. Thus, we propose that oncogene-containing tumor cell-derived MVs could act as a unique form of angiogenesis-modulating stimuli and are capable of switching endothelial cells to act in an autocrine mode.

Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells.

Aggressive human brain tumours (gliomas) often express a truncated and oncogenic form of the epidermal growth factor receptor, known as EGFRvIII. Within each tumour only a small percentage of glioma cells may actually express EGFRvIII; however, most of the cells exhibit a transformed phenotype. Here we show that EGFRvIII can be 'shared' between glioma cells by intercellular transfer of membrane-derived microvesicles ('oncosomes'). EGFRvIII expression in indolent glioma cells stimulates formation of lipid-raft related microvesicles containing EGFRvIII. Microvesicles containing this receptor are then released to cellular surroundings and blood of tumour-bearing mice, and can merge with the plasma membranes of cancer cells lacking EGFRvIII. This event leads to the transfer of oncogenic activity, including activation of transforming signalling pathways (MAPK and Akt), changes in expression of EGFRvIII-regulated genes (VEGF, Bcl-x(L), p27), morphological transformation and increase in anchorage-independent growth capacity. Thus, membrane microvesicles of cancer cells can contribute to a horizontal propagation of oncogenes and their associated transforming phenotype among subsets of cancer cells.

The role of tumor-and host-related tissue factor pools in oncogene-driven tumor progression.

Oncogenic events play an important role in cancer-related coagulopathy (Trousseau syndrome), angiogenesis and disease progression. This can, in part, be attributed to the up-regulation of tissue factor (TF) and release of TF-containing microvesicles into the pericellular milieu and the circulation. In addition, certain types of host cells (stromal cells, inflammatory cells, activated endothelium) may also express TF. At present, the relative contribution of host- vs tumor-related TF to tumor progression is not known. Our recent studies have indicated that the role of TF in tumor formation is complex and context-dependent. Genetic or pharmacological disruption of TF expression/activity in cancer cells leads to tumor growth inhibition in immunodeficient mice. This occurred even in the case of xenotransplants of human cancer cells, in which TF overexpression is driven by potent oncogenes (K-ras or EGFR). Interestingly, the expression of TF in vivo is not uniform and appears to be influenced by many factors, including the level of oncogenic transformation, tumor microenvironment, adhesion and the coexpression of markers of cancer stem cells (CSCs). Thus, minimally transformed, but tumorigenic embryonic stem (ES) cells were able to form malignant and angiogenic outgrowths in the absence of TF. However, these tumors were growth inhibited in hosts (mice) with dramatically reduced TF expression (low-TF mice). Depletion of host TF also resulted in changes affecting vascular patterning of some, but not all types of tumors. These observations suggest that TF may play different roles growth and angiogenesis of different tumors. Moreover, both tumor cell and host cell compartments may, in some circumstances, contribute to the functional TF pool. We postulate that activation of the coagulation system and TF signaling, may deliver growth-promoting stimuli (e.g. fibrin, thrombin, platelets) to dormant cancer stem cells (CSCs). Functionally, these influences may be tantamount to formation of a provisional (TF-dependent) cancer stem cell niche. As such these changes may contribute to the involvement of CSCs in tumor growth, angiogenesis and metastasis.