Extracellular vesicles released during hypoxia transport heparanase and enhance macrophage migration, endothelial tube formation and cancer cell stemness.

Heparanase is upregulated during the progression of most cancers and via its enzyme activity promotes extracellular matrix degradation, angiogenesis and cell migration. Heparanase expression is often associated with enhanced tumor aggressiveness and chemoresistance. We previously demonstrated that increased heparanase expression in tumor cells enhances secretion and alters the composition of tumor-released exosomes. In the present study, we discovered that extracellular vesicles (EVs) secreted by human multiple myeloma cells growing in hypoxic conditions exhibited elevated levels of heparanase cargo compared to EVs from cells growing in normoxic conditions. When macrophages (RAW 264.7 monocyte/macrophage-like cells) were exposed to EVs released by tumor cells growing in either hypoxic or normoxic conditions, macrophage migration and invasion was elevated by EVs from hypoxic conditions. The elevated invasion of macrophages was blocked by a monoclonal antibody that inhibits heparanase enzyme activity. Moreover, the heparanase-bearing EVs from hypoxic cells greatly enhanced endothelial cell tube formation consistent with the known role of heparanase in promoting angiogenesis. EVs from hypoxic tumor cells when compared with EVs from normoxic cells also enhanced cancer stemness properties of both CAG and RPMI 8226 human myeloma cells. Together these data indicate that under hypoxic conditions, tumor cells secrete EVs having an elevated level of heparanase as cargo. These EVs can act on both tumor and non-tumor cells, enhancing tumor progression and tumor cell stemness that likely supports chemoresistance and relapse of tumor.

Therapy-induced chemoexosomes: Sinister small extracellular vesicles that support tumor survival and progression.

Chemotherapy involves the use of multiple cytotoxic or cytostatic drugs acting by various mechanisms to kill or arrest the growth of cancer cells. Chemotherapy remains the most utilized approach for controlling cancer. Emerging evidence indicates that cancer cells activate various pro-survival mechanisms to cope with chemotherapeutic stress. These mechanisms persist during treatment and often help orchestrate tumor regrowth and patient relapse. Exosomes due to their nature of carrying and transferring multiple biologically active components have emerged as key players in cancer pathogenesis. Recent data demonstrates that chemotherapeutic stress enhances the secretion and alters the cargo carried by exosomes. These altered exosomes, which we refer to as chemoexosomes, are capable of transferring cargo to target tumor cells that can enhance their chemoresistance, increase their metastatic behavior and in certain cases even aid in endowing tumor cells with cancer stem cell-like properties. This mini-review summarizes the recent developments in our understanding of the impact chemoexosomes have on tumor survival and progression.

Heparanase-enhanced Shedding of Syndecan-1 and Its Role in Driving Disease Pathogenesis and Progression.

Both heparanase and syndecan-1 are known to be present and active in disease pathobiology. An important feature of syndecan-1 related to its role in pathologies is that it can be shed from the surface of cells as an intact ectodomain composed of the extracellular core protein and attached heparan sulfate and chondroitin sulfate chains. Shed syndecan-1 remains functional and impacts cell behavior both locally and distally from its cell of origin. Shedding of syndecan-1 is initiated by a variety of stimuli and accomplished predominantly by the action of matrix metalloproteinases. The accessibility of these proteases to the core protein of syndecan-1 is enhanced, and shedding facilitated, when the heparan sulfate chains of syndecan-1 have been shortened by the enzymatic activity of heparanase. Interestingly, heparanase also enhances shedding by upregulating the expression of matrix metalloproteinases. Recent studies have revealed that heparanase-induced syndecan-1 shedding contributes to the pathogenesis and progression of cancer and viral infection, as well as other septic and non-septic inflammatory states. This review discusses the heparanase/shed syndecan-1 axis in disease pathogenesis and progression, the potential of targeting this axis therapeutically, and the possibility that this axis is widespread and of influence in many diseases.

Heparanase promotes myeloma stemness and in vivo tumorigenesis.

Heparanase is known to enhance the progression of many cancer types and is associated with poor patient prognosis. We recently reported that after patients with multiple myeloma were treated with high dose chemotherapy, the tumor cells that emerged upon relapse expressed a much higher level of heparanase than was present prior to therapy. Because tumor cells having stemness properties are thought to seed tumor relapse, we investigated whether heparanase had a role in promoting myeloma stemness. When plated at low density and grown in serum-free conditions that support survival and expansion of stem-like cells, myeloma cells expressing a low level of heparanase formed tumor spheroids poorly. In contrast, cells expressing a high level of heparanase formed significantly more and larger spheroids than did the heparanase low cells. Importantly, heparanase-low expressing cells exhibited plasticity and were induced to exhibit stemness properties when exposed to recombinant heparanase or to exosomes that contained a high level of heparanase cargo. The spheroid-forming heparanase-high cells had elevated expression of GLI1, SOX2 and ALDH1A1, three genes known to be associated with myeloma stemness. Inhibitors that block the heparan sulfate degrading activity of heparanase significantly diminished spheroid formation and expression of stemness genes implying a direct role of the enzyme in regulating stemness. Blocking the NF-κB pathway inhibited spheroid formation and expression of stemness genes demonstrating a role for NF-κB in heparanase-mediated stemness. Myeloma cells made deficient in heparanase exhibited decreased stemness properties in vitro and when injected into mice they formed tumors poorly compared to the robust tumorigenic capacity of cells expressing higher levels of heparanase. These studies reveal for the first time a role for heparanase in promoting cancer stemness and provide new insight into its function in driving tumor progression and its association with poor prognosis in cancer patients.

Proteases and glycosidases on the surface of exosomes: Newly discovered mechanisms for extracellular remodeling.

Emergence of the field of exosome biology has opened an exciting door to better understand communication between cells. These tiny nanovesicles act as potent regulators of biological function by delivering proteins, lipids and nucleic acids from the cell of origin to target cells. Recently, several enzymes including membrane-type 1 matrix metalloproteinase (MT1-MMP), insulin-degrading enzyme (IDE), sialidase and heparanase, among others, were localized on the surface of exosomes secreted by various cell types. These exosomal surface enzymes retain their activity and can degrade their natural substrates present within extracellular spaces. To date, enzymes on exosome surfaces have been associated with the mobilization of growth factors, degradation of extracellular matrix macromolecules and destruction of amyloid ß plaques. This review focuses on the emerging role of exosomal surface enzymes and how this mechanism of remodeling within the extracellular space may regulate disease progression as related to cancer, inflammation and Alzheimer's disease.

Chemotherapy induces secretion of exosomes loaded with heparanase that degrades extracellular matrix and impacts tumor and host cell behavior.

The heparan sulfate-degrading enzyme heparanase promotes the progression of many cancers by driving tumor cell proliferation, metastasis and angiogenesis. Heparanase accomplishes this via multiple mechanisms including its recently described effect on enhancing biogenesis of tumor exosomes. Because we recently discovered that heparanase expression is upregulated in myeloma cells that survive chemotherapy, we were prompted to investigate the impact of anti-myeloma drugs on exosome biogenesis. When myeloma cells were exposed to the commonly utilized anti-myeloma drugs bortezomib, carfilzomib or melphalan, exosome secretion by the cells was dramatically enhanced. These chemotherapy-induced exosomes (chemoexosomes) have a proteome profile distinct from cells not exposed to drug including a dramatic elevation in the level of heparanase present as exosome cargo. The chemoexosome heparanase was not found inside the chemoexosome, but was present on the exosome surface where it was capable of degrading heparan sulfate embedded within an extracellular matrix. When exposed to myeloma cells, chemoexosomes transferred their heparanase cargo to those cells, enhancing their heparan sulfate degrading activity and leading to activation of ERK signaling and an increase in shedding of the syndecan-1 proteoglycan. Exposure of chemoexosomes to macrophages enhanced their secretion of TNF-α, an important myeloma growth factor. Moreover, chemoexosomes stimulated macrophage migration and this effect was blocked by H1023, a monoclonal antibody that inhibits heparanase enzymatic activity. These data suggest that anti-myeloma therapy ignites a burst of exosomes having a high level of heparanase that remodels extracellular matrix and alters tumor and host cell behaviors that likely contribute to chemoresistance and eventual patient relapse. SUMMARY: We find that anti-myeloma chemotherapy dramatically stimulates secretion of exosomes and alters exosome composition. Exosomes secreted during therapy contain high levels of heparanase on their surface that can degrade ECM and also can be transferred to both tumor and host cells, altering their behavior in ways that may enhance tumor survival and progression.

Chondroitin sulfate proteoglycan serglycin influences protein cargo loading and functions of tumor-derived exosomes.

Tumor cells produce and utilize exosomes to promote tumor growth and metastasis. Tumor-cell-derived exosomes deliver cargos that partially mimic the contents of the parent cell to nearby or distant normal or abnormal cells, thereby reprogramming the recipient cells to support tumor progression. Mechanisms by which tumor-derived exosomes subserve the tumor are under intense investigation. Here we demonstrate a critical role of the chondroitin sulfate proteoglycan serglycin in regulating the protein cargo and functions of myeloma cell-derived exosomes. Previous studies have shown that serglycin, the only known intracellular proteoglycan, functions mainly in the storage of basically charged components within the intracellular granules/vesicles via serglycin's densely clustered, negatively charged glycosaminoglycan chains. Here we demonstrate that serglycin plays a critical role in the protein cargo loading of tumor-derived exosomes. Serglycin was detected in exosomes derived from cell culture supernatants of human myeloma cell lines and serum of myeloma patients. Mass spectrometry analysis of exosomal proteins identified significantly fewer protein components within exosomes derived from serglycin-knockdown myeloma cells than within exosomes from control cells. On gene ontology analysis, exosomes derived from serglycin-knockdown cells, but not from control cells, lacked many proteins that are required for mediating different cellular processes. In functional assays, exosomes from serglycin-knockdown cells failed to induce an invasive phenotype in myeloma cells and failed to promote migration of macrophages. These findings reveal that serglycin plays an important role in maintaining the protein cargo in tumor-derived exosomes and suggest that targeting serglycin may temper the influence of these exosomes on cancer progression.