Heparanase Regulates Secretion,Composition, and Function of Tumor Cell-derived Exosomes

Emerging evidence indicates that exosomes play a key role in tumor-host cross-talk and that exosome secretion, composition, and functional capacity are altered as tumors progress to an aggressive phenotype. However, little is known regarding the mechanisms that regulate these changes. Heparanase is an enzyme whose expression is up-regulated as tumors become more aggressive and is associated with enhanced tumor growth, angiogenesis, and metastasis. We have discovered that in human cancer cells (myeloma, lymphoblastoid, and breast cancer), when expression of heparanase is enhanced or when tumor cells are exposed to exogenous heparanase, exosome secretion is dramatically increased. Heparanase enzyme activity is required for robust enhancement of exosome secretion because enzymatically inactive forms of heparanase, even when present in high amounts, do not dramatically increase exosome secretion. Heparanase also impacts exosome protein cargo as reflected by higher levels of syndecan-1, VEGF, and hepatocyte growth factor in exosomes secreted by heparanase-high expressing cells as compared with heparanase-low expressing cells. In functional assays, exosomes from heparanase-high cells stimulated spreading of tumor cells on fibronectin and invasion of endothelial cells through extracellular matrix better than did exosomes secreted by heparanase-low cells. These studies reveal that heparanase helps drive exosome secretion, alters exosome composition, and facilitates production of exosomes that impact both tumor and host cell behavior, thereby promoting tumor progression.     

M402, a novel heparan sulfate mimetic, targets multiple pathways implicated in tumor progression and metastasis

Heparan sulfate proteoglycans (HSPGs) play a key role in shaping the tumor microenvironment by presenting growth factors, cytokines, and other soluble factors that are critical for host cell recruitment and activation, as well as promoting tumor progression, metastasis, and survival. M402 is a rationally engineered, non-cytotoxic heparan sulfate (HS) mimetic, designed to inhibit multiple factors implicated in tumor-host cell interactions, including VEGF, FGF2, SDF-1α, P-selectin, and heparanase. A single s.c. dose of M402 effectively inhibited seeding of B16F10 murine melanoma cells to the lung in an experimental metastasis model. Fluorescent-labeled M402 demonstrated selective accumulation in the primary tumor. Immunohistological analyses of the primary tumor revealed a decrease in microvessel density in M402 treated animals, suggesting anti-angiogenesis to be one of the mechanisms involved in-vivo. M402 treatment also normalized circulating levels of myeloid derived suppressor cells in tumor bearing mice. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis and prolonged survival in an orthotopic 4T1 murine mammary carcinoma model. These data demonstrate that modulating HSPG biology represents a novel approach to target multiple factors involved in tumor progression and metastasis.     

Functional Characterization of Stromal Osteopontin in Melanoma Progression and Metastasis

Background

Recent studies demonstrated that not only tumor derived- but stroma derived factors play crucial role in cancer development. Osteopontin (OPN) is a secreted non-collagenous, sialic acid rich, chemokine-like phosphoglycoprotein that facilitates cell-matrix interactions and promotes tumor progression. Elevated level of OPN has been shown in melanoma patient and predicted as a prognostic marker. Recent reports have indicated that stroma-derived OPN are involved in regulating stem cell microenvironment and pre-neoplastic cell growth. However, the function of stroma derived OPN in regulation of side population (SP) enrichment leading to melanoma growth, angiogenesis and metastasis is not well studied and yet to be the focus of intense investigation.

Methodology/Principal Findings

In this study, using melanoma model, in wild type and OPN knockout mice, we have demonstrated that absence of host OPN effectively curbs melanoma growth, angiogenesis and metastasis. Melanoma cells isolated from tumor of OPN wild type (OPN^+/+) mice exhibited more tumorigenic feature as compared to the parental cell line or cells isolated from the tumors of OPN KO (OPN^−/−) mice. Furthermore, host OPN induces VEGF, ABCG2 and ERK1/2 expression and activation in B16-WT cells. We report for the first time that stroma derived OPN regulates SP phenotype in murine melanoma cells. Moreover, loss in and gain of function studies demonstrated that stroma-derived OPN regulates SP phenotype specifically through ERK2 activation.

Conclusions

This study establish at least in part, the molecular mechanism underlying the role of host OPN in melanoma growth and angiogenesis, and better understanding of host OPN-tumor interaction may assist the advancement of novel therapeutic strategy for the management of malignant melanoma.     

Heparanase enhances syndecan-1 shedding: a novel mechanism for stimulation of tumor growth and metastasis

When shed from the cell surface, the heparan sulfate proteoglycan syndecan-1 can facilitate the growth, angiogenesis, and metastasis of tumors. Here we report that tumor cell expression of heparanase, an enzyme known to be a potent promoter of tumor progression and metastasis, regulates both the level and location of syndecan-1 within the tumor microenvironment by enhancing its synthesis and subsequent shedding from the tumor cell surface. Heparanase regulation of syndecan-1 is detected in both human myeloma and breast cancer cell lines. This regulation requires the presence of active enzyme, because mutated forms of heparanase lacking heparan sulfate-degrading activity failed to influence syndecan-1 expression or shedding. Removal of heparan sulfate from the cell surface using bacterial heparitinase dramatically accelerated syndecan-1 shedding, suggesting that the effects of heparanase on syndecan-1 expression by tumor cells may be due, at least in part, to enzymatic removal or reduction in the size of heparan sulfate chains. Animals bearing tumors formed from cells expressing high levels of heparanase or animals transgenic for heparanase expression exhibited elevated levels of serum syndecan-1 as compared with controls, indicating that heparanase regulation of syndecan-1 expression and shedding can occur in vivo and impact cancer progression and perhaps other pathological states. These results reveal a new mechanism by which heparanase promotes an aggressive tumor phenotype and suggests that heparanase and syndecan-1 act synergistically to fine tune the tumor microenvironment and ensure robust tumor growth.     

Zinc depletion suppresses tumor growth in mice

The hypothesis that in tumor-bearing animals an increase of host hepatic zinc metallothionein (Zn-MT) causes a restriction of zinc in the tumor tissue was studied. Three types of tumors were induced in laboratory mice by cell transplant. Tumor growth appears to be inhibited under zinc-deficient conditions, even in cases where zinc deficiency was started after tumor cell transplant. The survival times of tumor-bearing mice were prolonged by administration of cadmium chloride, which induces the synthesis of a combined zinc-cadmium metallothionein derivative in the host liver, but not in the tumor tissue, leading to an increase of hepatic zinc in the treated animals. The uptake of⁶⁵Zn by the liver of Cd-treated, tumor bearing mice was significantly higher than that of controls whereas uptake of⁶⁵Zn by tumor cells was significantly higher in controls than in the treated animals. These results suggest that restriction of zinc intake suppresses tumor growth.     

Differential roles of type I interferon signaling in tumor versus host cells in experimental glioma models

Despite multimodal treatment approaches including surgery, radiotherapy and chemotherapy, the median survival for patients with glioblastoma remains in the range of one year and thus poor. Type I interferons (IFN) are involved in immune responses to viral infection and exhibit anti-tumor activity in certain cancers.Here we explored the biological relevance of constitutive type I IFN signaling in murine glioma models in vitro and in vivo. CT-2A, GL-261, SMA-497, SMA-540 and SMA-560 murine glioma cells expressed IFN type I receptors IFNAR1 and IFNAR2 and were responsive to exogenous IFN stimulation. CRISPR/Cas9-mediated deletion of IFNAR1 decreased the baseline expression of type I IFN response genes in GL-261 cells, but neither in CT-2A nor in SMA-560 cells. IFNAR1 deletion slowed growth in GL-261 and SMA-560, but not in CT-2A cells. However, only the growth of IFNAR1-depleted GL-261 tumors and not that of SMA-560 tumors was delayed in vivo upon orthotopic tumor cell implantation into syngeneic mice. This survival gain was no longer detected when the IFNAR1-depleted GL-261 cells were inoculated into IFNAR1-deficient mice. Altogether these data suggest that constitutive type I IFN signaling in gliomas may be pro-tumorigenic, but only in a microenvironment that is proficient for type I IFN signaling in the host.     

Suppression of tumorigenesis in mitochondrial NADP+-dependent isocitrate dehydrogenase knock-out mice

The tumor host microenvironment is increasingly viewed as an important contributor to tumor growth and suppression. Cellular oxidative stress resulting from high levels of reactive oxygen species (ROS) contributes to various processes involved in the development and progress of malignant tumors including carcinogenesis, aberrant growth, metastasis, and angiogenesis. In this regard, the stroma induces oxidative stress in adjacent tumor cells, and this in turn causes several changes in tumor cells including modulation of the redox status, inhibition of cell proliferation, and induction of apoptotic or necrotic cell death. Because the levels of ROS are determined by a balance between ROS generation and ROS detoxification, disruption of this system will result in increased or decreased ROS level. Recently, we demonstrated that the control of mitochondrial redox balance and cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) that supplies NADPH for antioxidant systems. To explore the interactions between tumor cells and the host, we evaluated tumorigenesis between IDH2-deficient (knock-out) and wild-type mice in which B16F10 melanoma cells had been implanted. Suppression of B16F10 cell tumorigenesis was reproducibly observed in the IDH2-deficient mice along with significant elevation of oxidative stress in both the tumor and the stroma. In addition, the expression of angiogenesis markers was significantly down-regulated in both the tumor and the stroma of the IDH2-deficient mice. These results support the hypothesis that redox status-associated changes in the host environment of tumor-bearing mice may contribute to cancer progression.     

Heparan sulfate proteoglycans and heparanase--partners in osteolytic tumor growth and metastasis.

This review summarizes a series of studies demonstrating that heparan sulfate proteoglycans act to promote the growth and metastasis of myeloma and breast tumors, two tumors that home to, and grow within, bone. Much of the growth-promoting effect of proteoglycans in these tumors may reside in the shed form of syndecan-1 that acts to favorably condition the tumor microenvironment. Moreover, the interplay between heparan sulfate and the extracellular enzyme heparanase-1 also has important regulatory implications. Recent studies indicate that the activity of heparanase, which likely releases heparin sulfate-bound growth factors and generates highly active heparan sulfate fragments, also promotes growth and metastasis of myeloma and breast tumors. Understanding the role of heparan sulfate and heparanase in the regulation of tumor behavior may lead to new therapeutic approaches for treating cancer.     

Heparanase Enhances the Insulin Receptor Signaling Pathway to Activate Extracellular Signal-regulated Kinase in Multiple Myeloma

ERK signaling regulates proliferation, survival, drug resistance, and angiogenesis in cancer. Although the mechanisms regulating ERK activation are not fully understood, we previously demonstrated that ERK phosphorylation is elevated by heparanase, an enzyme associated with aggressive behavior of many cancers. In the present study, myeloma cell lines expressing either high or low levels of heparanase were utilized to determine how heparanase stimulates ERK signaling. We discovered that the insulin receptor was abundant on cells expressing either high or low levels of heparanase, but the receptor was highly phosphorylated in heparanase-high cells compared with heparanase-low cells. In addition, protein kinase C activity was elevated in heparanase-high cells, and this enhanced expression of insulin receptor substrate-1 (IRS-1), the principle intracellular substrate for phosphorylation by the insulin receptor. Blocking insulin receptor function with antibody or a small molecule inhibitor or knockdown of IRS-1 expression using shRNA diminished heparanase-mediated ERK activation in the tumor cells. In addition, up-regulation of the insulin signaling pathway by heparanase and the resulting ERK activation were dependent on heparanase retaining its enzyme activity. These results reveal a novel mechanism whereby heparanase enhances activation of the insulin receptor signaling pathway leading to ERK activation and modulation of myeloma behavior.     

iNKT Cells Suppress the CD8(+) T Cell Response to a Murine Burkitt's-Like B Cell Lymphoma

The T cell response to B cell lymphomas differs from the majority of solid tumors in that the malignant cells themselves are derived from B lymphocytes, key players in immune response. B cell lymphomas are therefore well situated to manipulate their surrounding microenvironment to enhance tumor growth and minimize anti-tumor T cell responses. We analyzed the effect of T cells on the growth of a transplantable B cell lymphoma and found that iNKT cells suppressed the anti-tumor CD8(+) T cell response. Lymphoma cells transplanted into syngeneic wild type (WT) mice or Jalpha18(-/-) mice that specifically lack iNKT cells grew initially at the same rate, but only the mice lacking iNKT cells were able to reject the lymphoma. This effect was due to the enhanced activity of tumor-specific CD8(+) T cells in the absence of iNKT cells, and could be partially reversed by reconstitution of iNKT cells in Jalpha 18(-/-) mice. Treatment of tumor-bearing WT mice with alpha -galactosyl ceramide, an activating ligand for iNKT cells, reduced the number of tumor-specific CD8(+) T cells. In contrast, lymphoma growth in CD1d1(-/-) mice that lack both iNKT and type II NKT cells was similar to that in WT mice, suggesting that type II NKT cells are required for full activation of the anti-tumor immune response. This study reveals a tumor-promoting role for iNKT cells and suggests their capacity to inhibit the CD8(+) T cell response to B cell lymphoma by opposing the effects of type II NKT cells.     

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.     

Suppression of Natural Killer Cells by Sorafenib Contributes to Prometastatic Effects in Hepatocellular Carcinoma

Sorafenib, a multi-tyrosine kinase inhibitor, is a standard treatment for advanced hepatocellular carcinoma (HCC). The present study was undertaken to determine whether the growth and metastasis of HCC were influenced in mice receiving sorafenib prior to implantation with tumors, and to investigate the in-vivo and in-vitro effect of sorafenib on natural killer (NK) cells. In sorafenib-pretreated BALB/c nu/nu mice and C57BL/6 mice, tumor growth was accelerated, mouse survival was decreased, and lung metastasis was increased. However, the depletion of NK1.1⁺ cells in C57BL/6 mice eliminated sorafenib-mediated pro-metastatic effects. Sorafenib significantly reduced the number of NK cells and inhibited reactivity of NK cells against tumor cells, in both tumor-bearing and tumor-free C57BL/6 mice. Sorafenib down-regulated the stimulatory receptor CD69 in NK cells of tumor-bearing mice, but not in tumor-free mice, and inhibited proliferation of NK92-MI cells, which is associated with the blocking of the PI3K/AKT pathway, and inhibited cytotoxicity of NK cells in response to tumor targets, which was due to impaired ERK phosphorylation. These results suggest immunotherapeutic approaches activating NK cells may enhance the therapeutic efficacy of sorafenib in HCC patients.     

Decreased Peritoneal Ovarian Cancer Growth in Mice Lacking Expression of Lipid Phosphate Phosphohydrolase 1

Lysophosphatidic acid (LPA) is a bioactive lipid that enhances ovarian cancer cell proliferation, migration and invasion in vitro and stimulates peritoneal metastasis in vivo. LPA is generated through the action of autotaxin or phospholipases, and degradation begins with lipid phosphate phosphohydrolase (LPP)-dependent removal of the phosphate. While the effects of LPA on ovarian cancer progression are clear, the effects of LPA metabolism within the tumor microenvironment on peritoneal metastasis have not been reported. We examined the contribution of lipid phosphatase activity to ovarian cancer peritoneal metastasis using mice deficient in LPP1 expression. Homozygous deletion of LPP1 (LPP1 KO) results in elevated levels and decreased turnover of LPA in vivo. Within 2 weeks of intraperitoneal injection of syngeneic mouse ovarian cancer cells, we observed enhanced tumor seeding in the LPP1 KO mice compared to wild type. However, tumor growth plateaued in the LPP1 KO mice by 3 weeks while tumors continued to grow in wild type mice. The decreased tumor burden was accompanied by increased apoptosis and no change in proliferation or angiogenesis. Tumor growth was restored and apoptosis reversed with exogenous administration of LPA. Together, these observations demonstrate that the elevated levels of LPA per se in LPP1 KO mice do not inhibit tumor growth. Rather, the data support the notion that either elevated LPA concentration or altered LPA metabolism affects other growth-promoting contributions of the tumor microenvironment.     

GFP标记的肿瘤生长和转移的整体荧光成像

Fugene 6脂质体介导pEGFP-C1转染人源肺癌细胞(SPC-A1),经G418抗性筛选和96孔板有限稀释获得稳定高表达GFP的单克隆细胞株SPC-A1-EGFP。裸鼠腹腔注射SPC-A1-EGFP细胞建立自发转移模型;裸鼠尾静脉注射SPC-A1-EGFP细胞建立实验转移模型。利用整体光学成像系统(wllole-body optical imaging system)对荷瘤鼠整体荧光成像。结果表明,整体光学成像系统可实时非侵入监测腹腔肿瘤生长和扩散过程,通过胸腔皮瓣窗chest—wall skin-flap window)可低侵入检测肺转移。该研究为在体监测原位移植瘤的自发转移和发现抗肿瘤新药物提供了良好实验平台。     

The organ microenvironment and cancer metastasis

Primary neoplasms are biologically heterogeneous and the process of metastasis consists of a series of sequential, selective steps that few cells can complete. The outcome of cancer metastasis depends on multiple interactions between metastatic cells and homeostatic mechanisms that are unique to one or another organ microenvironment. The specific organ microenvironment determines the extent of cancer cell proliferation, angiogenesis, invasion, and survival. Therapy of metastasis should therefore be targeted not only against tumor cells, but also against the host factors that contribute to and support the progressive growth and survival of metastatic cancer cells.     

Identification of galectin-1 as a critical factor in function of mouse mesenchymal stromal cell-mediated tumor promotion

Bone marrow derived mesenchymal stromal cells (MSCs) have recently been implicated as one source of the tumor-associated stroma, which plays essential role in regulating tumor progression. In spite of the intensive research, the individual factors in MSCs controlling tumor progression have not been adequately defined. In the present study we have examined the role of galectin-1 (Gal-1), a protein highly expressed in tumors with poor prognosis, in MSCs in the course of tumor development. Co-transplantation of wild type MSCs with 4T1 mouse breast carcinoma cells enhances the incidence of palpable tumors, growth, vascularization and metastasis. It also reduces survival compared to animals treated with tumor cells alone or in combination with Gal-1 knockout MSCs. In vitro studies show that the absence of Gal-1 in MSCs does not affect the number of migrating MSCs toward the tumor cells, which is supported by the in vivo migration of intravenously injected MSCs into the tumor. Moreover, differentiation of endothelial cells into blood vessel-like structures strongly depends on the expression of Gal-1 in MSCs. Vital role of Gal-1 in MSCs has been further verified in Gal-1 knockout mice. By administering B16F10 melanoma cells into Gal-1 deficient animals, tumor growth is highly reduced compared to wild type animals. Nevertheless, co-injection of wild type but not Gal-1 deficient MSCs results in dramatic tumor growth and development.These results confirm that galectin-1 is one of the critical factors in MSCs regulating tumor progression.     

Direct Melanoma Cell Contact Induces Stromal Cell Autocrine Prostaglandin E2-EP4 Receptor Signaling That Drives Tumor Growth,Angiogenesis, and Metastasis

The stromal cells associated with tumors such as melanoma are significant determinants of tumor growth and metastasis. Using membrane-bound prostaglandin E synthase 1 (mPges1^−/−) mice, we show that prostaglandin E₂ (PGE₂) production by host tissues is critical for B16 melanoma growth, angiogenesis, and metastasis to both bone and soft tissues. Concomitant studies in vitro showed that PGE₂ production by fibroblasts is regulated by direct interaction with B16 cells. Autocrine activity of PGE₂ further regulates the production of angiogenic factors by fibroblasts, which are key to the vascularization of both primary and metastatic tumor growth. Similarly, cell-cell interactions between B16 cells and host osteoblasts modulate mPGES-1 activity and PGE₂ production by the osteoblasts. PGE₂, in turn, acts to stimulate receptor activator of NF-κB ligand expression, leading to osteoclast differentiation and bone erosion. Using eicosanoid receptor antagonists, we show that PGE₂ acts on osteoblasts and fibroblasts in the tumor microenvironment through the EP4 receptor. Metastatic tumor growth and vascularization in soft tissues was abrogated by an EP4 receptor antagonist. EP4-null Ptger4^−/− mice do not support B16 melanoma growth. In vitro, an EP4 receptor antagonist modulated PGE₂ effects on fibroblast production of angiogenic factors. Our data show that B16 melanoma cells directly influence host stromal cells to generate PGE₂ signals governing neoangiogenesis and metastatic growth in bone via osteoclast erosive activity as well as angiogenesis in soft tissue tumors.     

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.