Amos M. Sakwe

Detachment of Breast Tumor Cells Induces Rapid Secretion of Exosomes Which Subsequently Mediate Cellular Adhesion and Spreading

Exosomes are nano-vesicles secreted by a wide range of mammalian cell types. These vesicles are abundant in serum and other extracellular fluids and contain a large repertoire of proteins, mRNA and microRNA. Exosomes have been implicated in cell to cell communication, the transfer of infectious agents, and neurodegenerative diseases as well as tumor progression. However, the precise mechanisms by which they are internalized and/or secreted remain poorly understood. In order to follow their release and uptake in breast tumor cells in real time, cell-derived exosomes were tagged with green fluorescent protein (GFP)-CD63 while human serum exosomes were rhodamine isothiocynate-labeled. We show that detachment of adherent cells from various substrata induces a rapid and substantial secretion of exosomes, which then concentrate on the cell surfaces and mediate adhesion to various extracellular matrix proteins. We also demonstrate that disruption of lipid rafts with methyl-beta-cyclodextrin (MβCD) inhibits the internalization of exosomes and that annexins are essential for the exosomal uptake mechanisms. Taken together, these data suggest that cellular detachment is accompanied by significant release of exosomes while cellular adhesion and spreading are enhanced by rapid uptake and disposition of exosomes on the cell surface.

Anchorage-independent growth of breast carcinoma cells is mediated by serum exosomes

We hereby report studies that suggest a role for serum exosomes in the anchorage-independent growth (AIG) of tumor cells. In AIG assays, fetal bovine serum is one of the critical ingredients. We therefore purified exosomes from fetal bovine serum and examined their potential to promote growth of breast carcinoma cells in soft agar and Matrigel after reconstituting them into growth medium (EEM). In all the assays, viable colonies were formed only in the presence of exosomes. Some of the exosomal proteins we identified, have been documented by others and could be considered exosomal markers. Labeled purified exosomes were up-taken by the tumor cells, a process that could be competed out with excess unlabeled vesicles. Our data also suggested that once endocytosed by a cell, the exosomes could be recycled back to the conditioned medium from where they can be up-taken by other cells. We also demonstrated that low concentrations of exosomes activate MAP kinases, suggesting a mechanism by which they maintain the growth of the tumor cells in soft agar. Taken together, our data demonstrate that serum exosomes form a growth promoting platform for AIG of tumor cells and may open a new vista into cancer cell growth in vivo.

Annexin A6 contributes to the invasiveness of breast carcinoma cells by influencing the organization and localization of functional focal adhesions

The interaction of annexin A6 (AnxA6) with membrane phospholipids and either specific extracellular matrix (ECM) components or F-actin suggests that it may influence cellular processes associated with rapid plasma membrane reorganization such as cell adhesion and motility. Here, we examined the putative roles of AnxA6 in adhesion-related cellular processes that contribute to breast cancer progression. We show that breast cancer cells secrete annexins via the exosomal pathway and that the secreted annexins are predominantly cell surface-associated. Depletion of AnxA6 in the invasive BT-549 breast cancer cells is accompanied by enhanced anchorage-independent cell growth but cell-cell cohesion, cell adhesion/spreading onto collagen type IV or fetuin-A, cell motility and invasiveness were strongly inhibited. To explain the loss in adhesion/motility, we show that vinculin-based focal adhesions in the AnxA6-depleted BT-549 cells are elongated and randomly distributed. These focal contacts are also functionally defective because the activation of focal adhesion kinase and the phosphoinositide-3 kinase/Akt pathway were strongly inhibited while the MAP kinase pathway remained constitutively active. Compared with normal human breast tissues, reduced AnxA6 expression in breast carcinoma tissues correlates with enhanced cell proliferation. Together this suggests that reduced AnxA6 expression contributes to breast cancer progression by promoting the loss of functional cell-cell and/or cell-ECM contacts and anchorage-independent cell proliferation.

Lack of Fetuin-A (α2-HS-Glycoprotein) Reduces Mammary Tumor Incidence and Prolongs Tumor Latency via the Transforming Growth Factor-β Signaling Pathway in a Mouse Model of Breast Cancer

The present analyses were done to define the role of fetuin-A (Fet) in mammary tumorigenesis using the polyoma middle T antigen (PyMT) transgenic mouse model. We crossed Fet-null mice in the C57BL/6 background with PyMT mice in the same background and after a controlled breeding protocol obtained PyMT/Fet+/+, PyMT/Fet+/-, and PyMT/Fet-/- mice that were placed in control and experimental groups. Whereas the control group (PyMT/Fet+/+) formed mammary tumors 90 days after birth, tumor latency was prolonged in the PyMT/Fet-/- and PyMT/Fet+/- mice. The majority of the PyMT/Fet-/- mice were tumor-free at the end of the study, at approximately 40 weeks. The pathology of the mammary tumors in the Fet-null mice showed extensive fibrosis, necrosis, and squamous metaplasia. The preneoplastic mammary tissues of the PyMT/Fet-/- mice showed intense phopho-Smad2/3 staining relative to control tissues, indicating that transforming growth factor-β signaling is enhanced in these tissues in the absence of Fet. Likewise, p19ARF and p53 were highly expressed in tumor tissues of PyMT/Fet-/- mice relative to the controls in the absence of Fet. The phosphatidylinositol 3-kinase/Akt signaling pathway that we previously showed to be activated by Fet, on the other hand, was unaffected by the absence of Fet. The data indicate that Fet is a powerful modulator of breast tumorigenesis in this model system and has the potential to modulate breast cancer progression in humans.

Fetuin-A triggers the secretion of a novel set of exosomes in detached tumor cells that mediate their adhesion and spreading.

Our goal in this study was to define the mechanisms by which fetuin‐A mediates the adhesion of tumor cells. The data show that in the absence of fetuin‐A, detached tumor cells secrete exosomes that contain most of the known exosomal associated proteins but lack the capacity to mediate cellular adhesion. In the presence of fetuin‐A, the cells secrete exosomes, which contain, in addition to the other exosomal proteins, fetuin‐A, plasminogen and histones. These exosomes mediate adhesion and cell spreading. Plasminogen is a participant in this novel adhesion mechanism. The data suggest that these exosomes play a role in tumor progression.

Fetuin-A associates with histones intracellularly and shuttles them to exosomes to promote focal adhesion assembly resulting in rapid adhesion and spreading in breast carcinoma cells

The present analyses were undertaken to define the mechanisms by which fetuin-A modulates cellular adhesion. FLAG-tagged fetuin-A was expressed in breast carcinoma and HEK-293T cells. We demonstrated by confocal microscopy that fetuin-A co-localizes with histone H2A in the cell nucleus, forms stable complexes with histones such as H2A and H3 in solution, and shuttles histones to exosomes. The rate of cellular adhesion and spreading to either fibronectin or laminin coated wells was accelerated significantly in the presence of either endogenous fetuin-A or serum derived protein. More importantly, the formation of focal adhesion complexes on surfaces coated by laminin or fibronectin was accelerated in the presence of fetuin-A or histone coated exosomes. Cellular adhesion mediated by histone coated exosomes was abrogated by heparin and heparinase III. Heparinase III cleaves heparan sulfate from cell surface heparan sulfate proteoglycans. Lastly, the uptake of histone coated exosomes and subsequent cellular adhesion, was abrogated by heparin. Taken together, the data suggest a mechanism where fetuin-A, either endogenously synthesized or supplied extracellularly can extract histones from the nucleus or elsewhere in the cytosol/membrane and load them on cellular exosomes which then mediate adhesion by interacting with cell surface heparan sulfate proteoglycans via bound histones.

Reduced annexin A6 expression promotes the degradation of activated epidermal growth factor receptor and sensitizes invasive breast cancer cells to EGFR-targeted tyrosine kinase inhibitors

BackgroundThe expression of annexin A6 (AnxA6) in AnxA6-deficient non-invasive tumor cells has been shown to terminate epidermal growth factor receptor (EGFR) activation and downstream signaling. However, as a scaffolding protein, AnxA6 may stabilize activated cell-surface receptors to promote cellular processes such as tumor cell motility and invasiveness. In this study, we investigated the contribution of AnxA6 in the activity of EGFR in invasive breast cancer cells and examined whether the expression status of AnxA6 influences the response of these cells to EGFR-targeted tyrosine kinase inhibitors (TKIs) and/or patient survival.ResultsWe demonstrate that in invasive BT-549 breast cancer cells AnxA6 expression is required for sustained membrane localization of activated (phosho-Y1068) EGFR and consequently, persistent activation of MAP kinase ERK1/2 and phosphoinositide 3- kinase/Akt pathways. Depletion of AnxA6 in these cells was accompanied by rapid degradation of activated EGFR, attenuated downstream signaling and as expected enhanced anchorage-independent growth. Besides inhibition of cell motility and invasiveness, AnxA6-depleted cells were also more sensitive to the EGFR-targeted TKIs lapatinib and PD153035. We also provide evidence suggesting that reduced AnxA6 expression is associated with a better relapse-free survival but poorer distant metastasis-free and overall survival of basal-like breast cancer patients.ConclusionsTogether this demonstrates that the rapid degradation of activated EGFR in AnxA6-depleted invasive tumor cells underlies their sensitivity to EGFR-targeted TKIs and reduced motility. These data also suggest that AnxA6 expression status may be useful for the prediction of the survival and likelihood of basal-like breast cancer patients to respond to EGFR-targeted therapies.

Fetuin-A (α2HS-glycoprotein) is a serum chemo-attractant that also promotes invasion of tumor cells through Matrigel

The present study was conducted to determine whether fetuin-A, a dominant serum protein plays a role in chemo-attraction and chemo-invasion of carcinoma cells in vitro. Serum is normally used as positive chemotaxis control in Boyden chamber motility assays, prompting the need to identify the factor/s in serum that contributes the bulk of chemo-taxis and invasion. Serum has a plethora of chemotactic factors including stromal derived factor 1 also known as CXCL12. Using highly purified fetuin-A, we compared its chemo-attraction potential to culture medium containing 10% fetal bovine serum. We also investigated its ability to attract tumor cells through a bed of Matrigel (invasion assay). We demonstrated, using similar concentration range of fetuin-A found in blood, that it robustly supports both directed chemo-attraction and invasion of breast tumor cells. More importantly, we showed that at low concentrations (fetuin-A coated wells) itinteracts synergistically with CXCL12 to promote chemotaxis. The presence of plasminogen (PL) blunted the fetuin-A mediated chemotaxis. Taken together, the data suggest an in vivo chemotaxis/invasion role for fetuin-A.

Alpha-2 Heremans Schmid Glycoprotein (AHSG) Modulates Signaling Pathways in Head and Neck Squamous Cell Carcinoma Cell Line SQ20B

This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels of AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-β was examined to determine whether levels of the TGF-β binding AHSG influenced the effect of TGF-β on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-β influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis.

Human alpha defensin 5 is a candidate biomarker to delineate inflammatory bowel disease

Inability to distinguish Crohns colitis from ulcerative colitis leads to the diagnosis of indeterminate colitis. This greatly effects medical and surgical care of the patient because treatments for the two diseases vary. Approximately 30 percent of inflammatory bowel disease patients cannot be accurately diagnosed, increasing their risk of inappropriate treatment. We sought to determine whether transcriptomic patterns could be used to develop diagnostic biomarker(s) to delineate inflammatory bowel disease more accurately. Four patients groups were assessed via whole-transcriptome microarray, qPCR, Western blot, and immunohistochemistry for differential expression of Human α-Defensin-5. In addition, immunohistochemistry for Paneth cells and Lysozyme, a Paneth cell marker, was also performed. Aberrant expression of Human α-Defensin-5 levels using transcript, Western blot, and immunohistochemistry staining levels was significantly upregulated in Crohns colitis, p< 0.0001. Among patients with indeterminate colitis, Human α-Defensin-5 is a reliable differentiator with a positive predictive value of 96 percent. We also observed abundant ectopic crypt Paneth cells in all colectomy tissue samples of Crohns colitis patients. In a retrospective study, we show that Human α-Defensin-5 could be used in indeterminate colitis patients to determine if they have either ulcerative colitis (low levels of Human α-Defensin-5) or Crohns colitis (high levels of Human α-Defensin-5). Twenty of 67 patients (30 percent) who underwent restorative proctocolectomy for definitive ulcerative colitis were clinically changed to de novo Crohns disease. These patients were profiled by Human α-Defensin-5 immunohistochemistry. All patients tested strongly positive. In addition, we observed by both hematoxylin and eosin and Lysozyme staining, a large number of ectopic Paneth cells in the colonic crypt of Crohns colitis patient samples. Our experiments are the first to show that Human α-Defensin-5 is a potential candidate biomarker to molecularly differentiate Crohns colitis from ulcerative colitis, to our knowledge. These data give us both a potential diagnostic marker in Human α-Defensin-5 and insight to develop future mechanistic studies to better understand crypt biology in Crohns colitis.