Khalid Al-Nedawi

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.

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.

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.

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.

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 Peyers 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 hosts immune and nervous systems.—Al‐Nedawi, K., Mian, M. F., Hossain, N., Karimi, K., Mao, Y.‐K., Forsythe, P., Min, K. K., Stanisz, A. M., Kunze, W. A., Bienenstock, J. Gut commensal microvesicles reproduce parent bacterial signals to host immune and enteric nervous systems. FASEB J. 29, 684‐695 (2015). www.fasebj.org

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.

The Yin–Yang of Microvesicles (Exosomes) in Cancer Biology

Microvesicles and exosomes have emerged as a new mode of intercellular communication in cancer. In recent years, microvesicles have received increasing attention from the scientific community for their role in regulating and transferring active molecules responsible for tumor progression and metastasis. Controversy arises from the fact that microvesicles can transfer tumor-promoting molecules such as oncoproteins (1–3), and tumor suppressors (4, 5). So, how can we make sense of this controversy? By considering what we know of cancer development, we can rectify the seemingly opposing findings of the role of microvesicles in this process. The role of oncogenes in the progression of cancer is a well-investigated matter, and it is known that cancer progression is dependent on overexpression or mutation of oncogenic proteins. On the other hand, it also depends on loss or downregulation of tumor suppressor proteins. The effect of the intercellular exchange of oncoproteins and tumor suppressor proteins through microvesicles on cancer progression most probably follows the “Yin–Yang” concept. The availability of microvesicles rich in oncoproteins or those containing tumor suppressors will decide the new phenotype characteristics acquired by the acceptor cells. In the case of the tumor suppressor PTEN, it is interesting to note that metastases often have no expression of PTEN, although the primary tumor itself expresses PTEN (6). This may indicate that cells capable of initiating metastasis originate from cellular selection (7), whereby cells with little or no expression of PTEN are more likely to initiate successful metastases (8, 9). Furthermore, it is known from a series of studies of two tumor systems that tumors “talk” to each other through serum-borne factors (10–12). A tumor at one side of an experimental animal can affect the dynamics of a tumor on the other side. However, the serum-borne factors suggested in these studies are not fully defined; microvesicles represent a reasonable candidate as a player in this form of cellular communication. Such a mode of cellular communication may have a role in tumor metastasis. Microvesicles that contain PTEN are shed from primary PTEN-expressing tumors to the bloodstream, and may represent the serum-borne factors that are used by the primary tumor to affect the growth of metastases not expressing PTEN. This may indicate a successfully adopted mechanism throughout the natural history of cancer development, in which PTEN-null cells in the primary tumor form “tumor’s progeny” in the form of metastases. These metastases may be more aggressive than the primary tumor, because they do not express PTEN. The primary tumor then controls the growth of its “progeny” through tumor suppressor (PTEN) enriched microvesicles, which circulate in the blood and keep PTEN-null metastases in dormancy by supplementing them with PTEN protein. This hypothesis may explain the known phenomenon in which removal of the primary tumor enhances the growth of metastases, because the source of the PTEN-bearing microvesicles is removed. On the other hand, microvesicles enriched with oncogenic proteins may be shed by tumor cells to oppose the increased metabolic demand associated with overexpression of oncogenic proteins. Oncogenic protein expressing microvesicles are also used as intercellular mediators to engage other cells (which may be healthy) to provide a suitable growth niche to nourish cancer cells. For example, EGFR-bearing microvesicles stimulate endothelial cells to secrete vascular endothelial growth factor (VEGF) and the autophosphorylation of the VEGF receptor-2 (VEGFR2), triggering the angiogenic switch to initiate angiogenesis (1). It is noteworthy that microvesicles also contribute to the transfer of other molecules such as mRNAs and miRNA (miR), which can have various effects on tumor progression by modulating tumor microenvironment. An interesting cross-talk was found between hepatocarcinoma cells, whereby cells expressing miR-122 send this miR via microvesicles to inhibit the proliferation of miR-122 deficient cells. In a reciprocal process, miR-122 deficient cells secrete insulin-like growth factor to decrease miR-122 expression in miR-122 expressing cells (13). Microvesicles from cancer cells are found to stimulate tumor-associated macrophages to secrete VEGF, through the transfer of miR-150 (14) and thereby enhance tumorigenicity. In an opposite effect, macrophages inhibit proliferation of hepatocarcinoma cells by transferring miR-142 and miR-223 via microvesicles (15). A similar effect is found for microvesicles from marrow stromal cells expressing miR-146, which inhibit the proliferation of glioma cells (16). In this sense, the role of microvesicles that contain molecules whose effects are seemingly in opposition (oncoproteins, oncomirs, or tumor suppressors) may be reconciled from the perspective of the tumor, a successful parasitic organ generated from our own cells. We realize that this commentary may contain some speculations, but we hope that it can trigger serious discussions in tumor biology, which may contribute to the long battle against the killer known as cancer.

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/β-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 β-catenin mutants are identified in PCs leading to a rapid onset of death and recurrence. Genomic alterations in APC and CTNNB1/β-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.

Sterol Regulatory Element Binding Protein (SREBP)-1 is a novel regulator of the Transforming Growth Factor (TGF)-β receptor I (TβRI) 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-β1) 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-β1 as well as facilitation of its signaling. Here we have identified that SREBP-1 is also a novel regulator of TGF-β receptor I (TβRI) 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 TβRI transcription, nor did it induce its proteasomal or lysosomal degradation or proteolytic cleavage. Disruption of lipid rafts with cyclodextrin, however, prevented TβRI downregulation. This was not dependent on caveolae since SREBP-1 inhibition could induce TβRI downregulation in caveolin-1 knockout mesangial cells. SREBP-1 associated with TβRI, and SREBP-1 inhibition led to the secretion of TβRI in exosomes. Thus, we have identified a novel role for SREBP-1 as a cell surface retention factor for TβRI 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 TGFβ signaling and matrix upregulation.

Thrombotic characteristics of extracellular vesicles derived from prostate cancer cells.

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.