Veronica MacLeod

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.

Enzymatic remodeling of heparan sulfate proteoglycans within the tumor microenvironment: Growth regulation and the prospect of new cancer therapies

Heparan sulfate proteoglycans (HSPGs), via their interactions with numerous effector molecules such as FGF‐2, IL‐8, and VEGF, regulate the biological activity of cells by acting as co‐receptors that promote signaling. The extent and nature of their role as co‐receptors is often misregulated in cancer as manifested by alterations in HSPG structure and expression level. This misregulation of HSPGs can aid in promoting the malignant phenotype. In addition to expression‐related changes in HSPGs, recent discoveries indicate that HSPGs localized within the tumor microenvironment can be attacked by enzymes that alter proteoglycan structure resulting in dramatic effects on tumor growth and metastasis. This review focuses on remodeling of HSPGs by three distinct mechanisms that occur in vivo; (i) shedding of proteoglycan extracellular domains from cell surfaces, (ii) fragmentation of heparan sulfate chains by heparanase, and (iii) removal of sulfates from the 6‐O position of heparan sulfate chains by extracellular sulfatases. Assessing or monitoring the remodeling of HSPGs has important implications for tumor diagnosis and patient prognosis while therapeutic manipulation of the remodeling process represents an exciting new possibility for treating cancer. J. Cell. Biochem.

HSulf-1 and HSulf-2 Are Potent Inhibitors of Myeloma Tumor Growth in Vivo

To participate as co-receptor in growth factor signaling, heparan sulfate must have specific structural features. Recent studies show that when the levels of 6-O-sulfation of heparan sulfate are diminished by the activity of extracellular heparan sulfate 6-O-endosulfatases (Sulfs), fibroblast growth factor 2-, heparin binding epidermal growth factor-, and hepatocyte growth factor-mediated signaling are attenuated. This represents a novel mechanism for regulating cell growth, particularly within the tumor microenvironment where the Sulfs are known to be misregulated. To directly test the role of Sulfs in tumor growth control in vivo, a human myeloma cell line was transfected with cDNAs encoding either of the two known human endosulfatases, HSulf-1 or HSulf-2. When implanted into severe combined immunodeficient (SCID) mice, the growth of these tumors was dramatically reduced on the order of 5- to 10-fold as compared with controls. In addition to an inhibition of tumor growth, these studies revealed the following. (i) HSulf-1 and HSulf-2 have similar functions in vivo. (ii) The extracellular activity of Sulfs is restricted to the local tumor cell surface. (iii) The Sulfs promote a marked increase in extracellular matrix deposition within tumors that may, along with attenuated growth factor signaling, contribute to the reduction in tumor growth. These findings demonstrate that dynamic regulation of heparan sulfate structure by Sulfs present within the tumor microenvironment can have a dramatic impact on the growth and progression of malignant cells in vivo.

Expression of Heparanase by Primary Breast Tumors Promotes Bone Resorption in the Absence of Detectable Bone Metastases

Heparanase is an enzyme that cleaves heparan sulfate and through this activity promotes tumor growth, angiogenesis, invasion, and metastasis in several tumor types. In human breast cancer patients, heparanase expression is associated with sentinel lymph node metastases. However, the precise role of heparanase in the malignant progression of breast cancer is unknown. To examine this, a variant of MDA-MB-231 cells was transfected with the cDNA for human heparanase (HPSE cells) or with vector alone as a control (NEO cells). Transfection produced a 6-fold increase in heparanase activity in HPSE cells relative to NEO cells. When injected into the mammary fat pads of severe combined immunodeficient mice, the tumors formed by HPSE cells initially grow significantly faster than the tumors formed by NEO cells. The rapid growth is due in part to increased angiogenesis, as microvessel densities are substantially elevated in primary HPSE tumors compared with NEO tumors. Although metastases to bones are not detected, surprisingly vigorous bone resorption is stimulated in animals bearing tumors formed by the HPSE cells. These animals have high serum levels of the C-telopeptide derived from type I collagen as well as significant elevation of the active form of tartrate-resistant acid phosphatase (TRAP)-5b. In contrast, in animals having a high tumor burden of Neo cells, the serum levels of C-telopeptide and TRAP-5b never increase above the levels found before tumor injection. Consistent with these findings, histologic analysis for TRAP-expressing cells reveals extensive osteoclastogenesis in animals harboring HPSE tumors. In vitro osteoclastogenesis assays show that the osteoclastogenic activity of HPSE cell conditioned medium is significantly enhanced beyond that of NEO conditioned medium. This confirms that a soluble factor or factors that stimulate osteoclastogenesis are specifically produced when heparanase expression is elevated. These factors exert a distal effect resulting in resorption of bone and the accompanying enrichment of the bone microenvironment with growth-promoting factors that may nurture the growth of metastatic tumor cells. This novel role for heparanase as a promoter of osteolysis before tumor metastasis suggests that therapies designed to block heparanase function may disrupt the early progression of bone-homing tumors.

Syndecan-1 Is Required for Robust Growth, Vascularization, and Metastasis of Myeloma Tumors in Vivo

Myeloma tumors are characterized by high expression of syndecan-1 (CD138), a heparan sulfate proteoglycan present on the myeloma cell surface and shed into the tumor microenvironment. High levels of shed syndecan-1 in the serum of patients are an indicator of poor prognosis, and numerous studies have implicated syndecan-1 in promoting the growth and progression of this cancer. In the present study we directly addressed the role of syndecan-1 in myeloma by stable knockdown of its expression using RNA interference. Knockdown cells that were negative for syndecan-1 expression became apoptotic and failed to grow in vitro. Knockdown cells expressing syndecan-1 at ∼28% or ∼14% of normal levels survived and grew well in vitro but formed fewer and much smaller subcutaneous tumors in mice compared with tumors formed by cells expressing normal levels of syndecan-1. When injected intravenously into mice (experimental metastasis model), knockdown cells formed very few metastases as compared with controls. This indicates that syndecan-1 may be required for the establishment of multi-focal metastasis, a hallmark of this cancer. One mechanism of syndecan-1 action occurs via stimulation of tumor angiogenesis because tumors formed by knockdown cells exhibited diminished levels of vascular endothelial growth factor and impaired development of blood vessels. Together, these data indicate that the effects of syndecan-1 on myeloma survival, growth, and dissemination are due, at least in part, to its positive regulation of tumor-host interactions that generate an environment capable of sustaining robust tumor growth.

Molecular Cloning and Functional Characterization of a Vasotocin Receptor Subtype That Is Expressed in the Shell Gland and Brain of the Domestic Chicken

Abstract In chickens, oviposition is correlated with increased plasma levels of the neurohypophysial hormone vasotocin, and vasotocin stimulates contraction of uterine strips in vitro. A gene encoding a vasotocin receptor subtype that we have designated the VT1 receptor was cloned from the domestic chicken. The open reading frame encodes a 370-amino acid polypeptide that displays seven segments of hydrophobic amino acids, typical of guanine nucleotide-protein-coupled receptors. Other structural features of the VT1 receptor include two potential N-linked glycosylation sites in the extracellular N-terminal region, a conserved aspartic acid in transmembrane domain 2 that is found in nearly all guanine nucleotide-protein-coupled receptors, and two potential protein kinase C phosphorylation sites in the third intracellular loop and C-terminal tail. Expressed VT1 receptors in COS7 cells bind neurohypophysial hormones with the following rank order of potency: vasotocin ≅ vasopressin > oxytocin ≅ mesotocin > isotocin. In addition, the expressed VT1 receptor mediates vasotocin-induced phosphatidylinositol turnover and Ca2+ mobilization. In the chicken, expression of VT1 receptor gene transcripts is limited to the shell gland (uterus) and the brain. Thus, the VT1 receptor that we have cloned may mediate contractions of the shell gland during oviposition and activate reproductive behaviors known to be stimulated by vasotocin in lower vertebrates.

Tumor-derived syndecan-1 mediates distal cross-talk with bone that enhances osteoclastogenesis.

Tumor‐stimulated bone resorption fuels tumor growth and marks a dramatic decline in the health and prognosis of breast cancer patients. Identifying mechanisms that mediate cross‐talk between tumor and bone remains a key challenge. We previously demonstrated that breast cancer cells expressing high levels of heparanase exhibit enhanced shedding of the syndecan‐1 proteoglycan. Moreover, when these heparanase‐high cells are implanted in the mammary fat pad, they elevate bone resorption. In this study, conditioned medium from breast cancer cells expressing high levels of heparanase was shown to significantly stimulate human osteoclastogenesis in vitro (p < .05). The osteoclastogenic activity in the medium of heparanase‐high cells was traced to the presence of syndecan‐1, intact heparan sulfate chains, and heat‐labile factor(s), including the chemokine interleukin 8 (IL‐8). The enhanced osteoclastogenesis promoted by the heparanase‐high cells results in a dramatic increase in bone resorption in vitro. In addition, the long bones of animals bearing heparanase‐high tumors in the mammary fat pad had significantly higher numbers of osteoclasts compared with animals bearing tumors expressing low levels of heparanase (p < .05). Together these data suggest that syndecan‐1 shed by tumor cells exerts biologic effects distal to the primary tumor and that it participates in driving osteoclastogenesis and the resulting bone destruction.

Amplification of JNK Signaling Is Necessary To Complete the Murine Gammaherpesvirus 68 Lytic Replication Cycle

ABSTRACT Several studies have previously defined host-derived signaling events capable of driving lytic gammaherpesvirus replication or enhancing immediate-early viral gene expression. Yet signaling pathways that regulate later stages of the productive gammaherpesvirus replication cycle are still poorly defined. In this study, we utilized a mass spectrometric approach to identify c-Jun as an abundant cellular phosphoprotein present in late stages of lytic murine gammaherpesvirus 68 (MHV68) infection. Kinetically, c-Jun phosphorylation was enhanced as infection progressed, and this correlated with enhanced phosphorylation of the c-Jun amino-terminal kinases JNK1 and JNK2 and activation of AP-1 transcription. These events were dependent on progression beyond viral immediate-early gene expression, but not dependent on viral DNA replication. Both pharmacologic and dominant-negative blockade of JNK1/2 activity inhibited viral replication, and this correlated with inhibition of viral DNA synthesis and reduced viral gene expression. These data suggest a model in which MHV68 by necessity amplifies and usurps JNK/c-Jun signaling as infection progresses in order to facilitate late stages of the MHV68 lytic infection cycle.

Phosphoproteomic analyses reveal signaling pathways that facilitate lytic gammaherpesvirus replication.

Lytic gammaherpesvirus (GHV) replication facilitates the establishment of lifelong latent infection, which places the infected host at risk for numerous cancers. As obligate intracellular parasites, GHVs must control and usurp cellular signaling pathways in order to successfully replicate, disseminate to stable latency reservoirs in the host, and prevent immune-mediated clearance. To facilitate a systems-level understanding of phosphorylation-dependent signaling events directed by GHVs during lytic replication, we utilized label-free quantitative mass spectrometry to interrogate the lytic replication cycle of murine gammaherpesvirus-68 (MHV68). Compared to controls, MHV68 infection regulated by 2-fold or greater ca. 86% of identified phosphopeptides – a regulatory scale not previously observed in phosphoproteomic evaluations of discrete signal-inducing stimuli. Network analyses demonstrated that the infection-associated induction or repression of specific cellular proteins globally altered the flow of information through the host phosphoprotein network, yielding major changes to functional protein clusters and ontologically associated proteins. A series of orthogonal bioinformatics analyses revealed that MAPK and CDK-related signaling events were overrepresented in the infection-associated phosphoproteome and identified 155 host proteins, such as the transcription factor c-Jun, as putative downstream targets. Importantly, functional tests of bioinformatics-based predictions confirmed ERK1/2 and CDK1/2 as kinases that facilitate MHV68 replication and also demonstrated the importance of c-Jun. Finally, a transposon-mutant virus screen identified the MHV68 cyclin D ortholog as a viral protein that contributes to the prominent MAPK/CDK signature of the infection-associated phosphoproteome. Together, these analyses enhance an understanding of how GHVs reorganize and usurp intracellular signaling networks to facilitate infection and replication.

Abstract 3516: EZH2 overexpression in myeloma patients shortens survival and in-vitro data supports a potential new targeted treatment strategy

Enhancer of zeste homolog 2 (EZH2) induces methylation at histone 3 lysine 27 (H3K27), thought to have a repressive effect on target gene expression. Activating mutations in EZH2 have been shown to drive lymphomas and overexpression linked to poor prognosis in solid tumors. In normal plasma cell development EZH2 expression is downregulated on exit from the germinal centre but in myeloma (MM) expression has been shown to increase with disease progression. Recent work suggests high expression of the H3K36 methyltransferase MMSET sensitizes cells to EZH2 inhibition. We are seeking to further investigate the role of EZH2 in MM. Data were analyzed from newly diagnosed MM patients in several large clinical trials (Whole-exome sequencing, n = 463 UK Myeloma XI, Affymetrix gene expression array (GEP), n = 259 UK Myeloma IX and n = 1213 UAMS Total Therapy Protocols). Across the datasets, high expression of EZH2 mRNA shortened patient survival (e.g. UK IX median OS, 29.9 months vs 45.1 months, Log-rank p = 0.005), remaining significant on multivariate analyses and across GEP defined molecular subgroups. Of note, the proliferative subgroup had a higher mean expression of EZH2 (9.56 vs 8.48, t-test p = 5.953e-42) as did high risk patients defined by GEP 70 score (9.61 vs 8.46, t-test p = 1.993e-40) suggesting a link to a more aggressive disease phenotype. There were no mutations in EZH2. This contrasts with frequent mutations seen in malignancies at earlier stages of B cell development e.g. DLBCL. Of note, 3% of MM patients had a potentially inactivating mutation or deletion in the gene encoding the H3K27 demethylase, KDM6A and significantly shorter OS than those without (% alive 2 yr: Mutated 51% (CI 30-85) vs 80% (CI 77-84), Log-rank p = 0.0498). Such mutations may increase H3K27 methylation, potentially sensitizing patients to EZH2 inhibition. To study the biological implications of our results we tested the effect of inhibition of EZH2 (EZH2i), using a small molecule EPZ005687, in a panel of MM cell lines representing the epi/genetic diversity of MM. We saw a reduction in total cell number and% viability at 6 days of EZH2i (2-4uM) in 5/8 cell lines with evidence of dose dependent induction of apoptosis. All cell lines (except LP1) had similar levels of EZH2 protein at baseline and the responding cell lines had no unifying epi/genetic anomalies, suggesting response is not specific to either MMSET high or KDM6A mut/del cell lines. 6 days of EZH2i reduced H3K27me2/me3 on Western blotting in all cell lines regardless of viability response, demonstrating the epigenetic activity of the inhibitor. Further evaluation by Chip-seq and GEP is ongoing to identify potential differences, e.g. in focal methylation marks, that may account for differing viability responses. Our results highlight the prognostic significance of EZH2 in MM and demonstrate excellent in-vitro inhibitor activity suggesting EZH2 as a possible future target for treatment. Citation Format: Charlotte Pawlyn, Martin F. Kaiser, Caleb K. Stein, Christopher P. Wardell, Veronica Macleod, Rick Edmondson, Bart Barlogie, Brian Walker, Gareth J. Morgan, Faith E. Davies. EZH2 overexpression in myeloma patients shortens survival and in-vitro data supports a potential new targeted treatment strategy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3516. doi:10.1158/1538-7445.AM2015-3516