Alan C. Rapraeger, PhD, University of California Berkeley 1979 - US grants

DESCRIPTION (provided by applicant): The syndecans are a family of heparan sulfate proteoglycans that function as receptors in extracellular matrix adhesion and growth factor signaling. Of the four family members, syndecan-1 is the most abundant on epithelial cells. The core protein of this receptor has a highly conserved but short cytoplasmic domain, a conserved transmembrane domain, and a more highly variable extracellular )rotein domain (ectodomain). Our recent work has identified several novel interactions of this core protein, showing that each of these discrete domains has unique activity. We plan to examine the unique activities that we have described for the cytoplasmic domain and extracellular domain in human nammary epithelial cells. We will examine the newly discovered role of the syndecan-1 ectodomain in activation of the ava3 integrin. This integrin is expressed on a number of invasive mammary carcinomas and its activated form leads to mammary carinoma cell metastases. We will attempt to define how syndecan-1 regulates the activity of this receptor, and define inhibitors to block its actvity. We will also examine the role of syndecan-1 in the activity of alphavbeta4 integrin. This integrin is critical for the normal polarized phenotype of mammary epithelial cells and their anchorage to their underlying basal lamina ich in laminin 5. However, the integrin is converted to a motility-signaling role on invasive cells. We wil examine how syndecan-1 participates in activity and thus contributes to the tumorigenic phenotype of transformed cells. Finally, we will assess normal and tumorigenic mammary epithelial cells in a three-dimensional matrigel culture, in which the cells display a phenotype more similar to that observed in vivo, and rely on signaling mechanisms differently that in common two-dimensional experiments. We will examine the role of syndecan-1 and the seemingly dramatic role that it has on human mammary epithelial cells in this environment. We will then extend these studies to a tumorigenesis model using the human carcinoma cells in SCID mice. We anticipate that completion of these studies will provide new and unexpected insights into the function of this receptor, and will lead to the development of new cancer treatments.

The heparin binding growth factors (fibroblast growth factor or FGFs) are a family of polypeptides that regulate a variety of cellular behaviors, including cell proliferation, migration and shape change, and cellular determination and differentiation. The diversity of these responses suggests intricate signalling mechanisms. One of the intricacies that will be studied here is the requirement for two types of FGF binding molecules to collaborate at the cell surface in order for FGF signalling to be carried out. One molecule is a membrane-spanning receptor with a cytoplasmic tyrosine-kinase domain that is activated upon ligand binding. The second molecule is a heparan sulfate proteoglycan that binds FGF via its heparan sulfate chains. This latter class of molecules is typified by syndecan, a transmembrane protein. Several other proteoglycans of this general type have been described. A contrasting form of cell surface proteoglycan is glypican, which is tethered to the membrane by a lipid tail. The aim of this work will be to understand the biochemistry of how these types of proteoglycans interact with the tyrosine kinase receptors in order to regulate FGF binding and signalling. Potential regulatory mechanisms include (i) the specific structure of the heparan sulfate chains, which are known to exhibit widely differing affinities for the growth factor, affinities that may vary between the two types of proteoglycans and are expected to vary among cell types and (ii) the nature of the proteoglycan anchorage in the membrane, which may be a critical regulator in the formation of an active complex between the proteoglycan, the receptor and the FGF. These regulatory mechanisms will be studied using two well-defined system: (i) a lymphoid cell expressing a single FGF receptors or receptors and proteoglycans expressed as pairs, and (ii) a myoblast cell line that responds quantitatively to FGF. The lymphoid cells will provide valuable information on ow a single type of receptor interacts with a single type of proteoglycan. The myoblast will provide the means for verifying the role of the interaction in FGF signalling. The potent role of the FGFs in cell growth and differentiation makes them obvious candidates for defects that lead to human disease, particularly in cancer, birth defects and neuromuscular degenerative disorders. Specific examples include their induction of mammary carcinomas, role in sarcoma growth or role as a tumor angiogenic factor. A better understanding of how FGF action is regulated in these diseases will provide insights into potential treatments.

The fibroblast growth factors (FGFs) are a family of 22 related polypeptides that regulate the proliferation, differentiation and migration of a wide variety of cell types. FGF1 and FGF2 are the prototypes of the family initially described as promoting neuronal proliferation, neurite extension and survival, and fibroblast growth, respectively. Many of their native roles are in early development, where they control early aspects of embryonic differentiation. Thus, an understanding of how these growth factors signal, and how this signaling may be regulated or controlled, will have direct applicability to the treatment of cancer and birth defects. One of the intricacies of FGF signaling is that two types of receptors collaborate at the cell surface to generate the signal. One of these types is a family of receptor tyrosine kinases The other type if heparan sulfate proteoglycans. The FGFs are united by a common affinity for haparan sulfate, the highly sulfated glycosaminoglycan chain found on heparan sulfate proteogycans of the cell surface and the extracellular matrix. In addition, the receptors have a heparan sulfate- binding domain, and successful docking of the FGF with the receptor relies on both proteins recognizing a common domain in the heparan sulfate chain. An important feature of heparan sulfate is its variable sulfation patterns. Our hypothesis, for which we now have evidence, is that this sulfation pattern may be cell- or tissue-type specific. Thus, although it is variable, it is not random. This proposal will use FGF and FGF receptor probes to detect structural differences in heparan sulfate during early mouse development. The probes will be used to isolate the specific heparan sulfate sequences to which they bind, and these sequences will then be identified chemically. The long term outcome of this work will be left the derivation of chemical mimetics that can be used as therapeutics in the treatment of cancer and the prevention of birth defects.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The 12th Gordon Conference on Proteoglycans will be held July 9th-14th at Proctor Academy in Andover, New Hampshire, USA. The purpose of this meeting will be to bring together members of the proteoglycan research community from around the world to present and discuss the most recent discoveries in the structure, function, and genetics of proteoglycans, with particular focus on signaling in disease mechanisms. The conference will continue the leading role that it has played over the past 20 years in serving as a catalyst for growth in this research community. The proteoglycan research group includes worldwide experts in chemical, cell biological, biochemical, engineering and clinical areas from academic, government and corporate laboratories. The major themes to be covered in the program include 1) Biosynthesis and Structure of Glycosaminoglycans, 2) Structure-Activity of Glycosaminoglycans in Growth Factor and Chemokine Signaling, 3) Proteoglycan Signaling in the Musculoskeletal System, 4) Proteoglycans in Cell Signaling Mechanisms, 5) Proteoglycans in Development, 6) Proteoglycans in Injury and Inflammation, 7) Proteoglycans in Cancer, and 8) Proteoglycans in Disease Models. We have planned nine sessions with a total of 31 plenary talks, with each session chaired by a known expert in the field. The speakers for the plenary talks are chosen with the primary goal of representing the top recent findings in the field. Additionally, the conference will encourage the participation of "new faces," namely, experts from other fields or disease specialties that have discovered new roles for proteoglycans in their area of expertise, or are speakers that are newly emerging on the international stage with new strategies or insights. In addition, another 20-25 short talks will be selected from the submitted abstracts, providing speaking opportunities for conferees with new and exciting findings, to provide opportunities for newer and underrepresented investigators, and to cover topics underrepresented in the plenary talks. The talks will be supplemented by active poster sessions and discussions. In all, investigators participating in this conference will be at all levels, including investigators currently funded by NIAMS, NCI, NIDCR, NINDS, NHLBI, NIA, NIDDK, NICHD, NIGMS, NCCR and other NIH Institutes. As has been the tradition at this conference, approximately a third of the planned presentations will report novel data on the role of proteoglycans in mechanisms relevant to inflammation and arthritis and other diseases of the musculoskeletal system and bone; major emphasis will also be given to proteoglycans in the cell biology of cancer invasion and tumor angiogenesis, and diseases of the vascular system and heart, and kidney, brain, and skin. Emphasis will also be given to proteoglycan signaling mechanisms and organ development. The culmination of the meeting will be two talks focused on new methodologies of discovery and approaches for new drug design based on newly discovered mechanisms. The goal of the conference is to promote the exchange of new findings, concepts and technology related to proteoglycan research into human disease. It is anticipated that the outcome of the conference will be an enhanced understanding of numerous human diseases, including those of human development, the musculoskeletal system, nervous system, vascular system, immunity and cancer, and that this understanding will ultimately lead to the formulation of new drugs for combating these diseases. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Syndecan-1 is one of a family of heparan sulfate proteoglycans that function as receptors in extracellular matrix adhesion. Although syndecan-1 is reportedly not expressed on endothelial cells lining mature vessels, we have found that it is expressed on activated endothelial cells undergoing tumor-induced angiogenesis and propose that it regulates the activity of the alpha-v-beta3 and alpha-v-beta5 integrins at this site; these two integrins are integral to the pathways by which FGF and VEGF induce angiogenesis, particularly providing protection against apoptosis. Our recent work has identified a novel mechanism in which syndecan-1 regulates the activity of the alpha-v-beta3 and alpha-v-beta5 integrins. Our preliminary findings suggest that silencing of syndecan-1 expression, or competition with the syndecan-1 ectodomain expressed in bacteria, will block the activation of these two integrins in endothelial cells. We propose to localize the site(s) responsible for regulating these integrin (s) and to isolate peptides representing the active sites in syndecan-1 that can be used competitively in angiogenesis assays. We also plan to molecularly characterize the regulatory complex that encompasses the syndecan and each integrin. Since syndecan-1 appears upregulated in vascular endothelium undergoing tumor-induced angiogenesis, we will explore what regulates this expression during the angiogenesis program, comparing stimulation by FGF and VEGF, and correlating syndecan-1 expression with the Hox D3, B3 and DIG master regulatory genes. These assays will also measure the two different downstream pathways of protection against apoptosis activated by either FGF or VEGF, as they have been shown to depend on the alpha-v-beta3 and alpha-v-beta5 integrins, respectively, and determine whether both rely on the syndecan-1-mediated regulation of these integrins. Finally, we will use this information, and the competitive peptides that we hope to derive, to disrupt the syndecan-1 regulation in vivo, using a mouse cornea angiogenesis assay, and angiogenesis induced by mouse tumors. These studies will provide new insights into how syndecan-1 functions during angiogenesis, and will hopefully lead to new drugs that block the deleterious angiogenesis that occurs in diseases such as cancer, endometriosis, diabetic retinopathy and others. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Multiple myeloma is the second most prevalent hematologic malignancy and accounts for over 10% of all hematologic cancers in the United States. It is estimated that over 16,500 new cases of myeloma are diagnosed and over 11,000 die from this disease each year. Although progress has been made in treatment over the last decade, the overall outlook for patients is grim. Multiple myeloma is a disease in which malignant plasma cells invade to populate and form tumors within the bone marrow. Their interactions with the tumor microenvironment lead to the release of cytokines that support myeloma cell proliferation, stimulate angiogenesis that supports myeloma cell growth and metastasis, and trigger bone lytic disease by over-stimulating differentiation of osteoclasts and their ensuing destruction of the bone. We have discovered a central mechanism in all of these processes that involves four effectors, each known to have a role in myeloma formation and progression - namely, the matrix receptor syndecan-1 (Sdc1), the av[unreadable]3 and av[unreadable]3 integrins, the insulin-like growth factor-1 receptor, and heparanase. The central mechanism is activation of a signaling complex comprised of Sdc1, the two integrins and the IGF1R when Sdc1 is activated by cleavage of its heparan sulfate chains by heparanase. Importantly, this mechanism is blocked by a peptide (called synstatin (SSTN)) that targets the active site on syndecan-1. This proposal will examine the mechanism by which Sdc1 is activated by heparanase on the myeloma cells, leading to tumorigenesis of the myeloma and heightened activation of vascular endothelial cells and osteoclast progenitors in the tumor microenvironment. Next, we will test the efficacy of SSTN as an inhibitor of this mechanism on the tumor cells and the cells in their microenvironment. The benefit of this work is likely to be new and effective treatments for multiple myeloma and other cancers. PUBLIC HEALTH RELEVANCE: Multiple myeloma is the second most prevalent hematologic malignancy. A novel mechanism, based on the matrix receptor syndecan-1 that is highly expressed in multiple myeloma, will be targeted by a new therapeutic called synstatin. Synstatin will be tested for its efficacy on the myeloma tumor and on the tumor microenvironment.

DESCRIPTION (provided by applicant): The ?4 subunit of the ?6?4 integrin, which forms hemidesmosomes in quiescent normal cells, becomes phosphorylated in breast tumor cells that overexpress HER2 or EGFR. This phosphorylation converts the cytoplasmic domain of the integrin into a signaling scaffold that drives cell invasion, proliferation and survival. HER2 and EGFR are expressed in HER2+/ER- breast cancer, and EGFR is overexpressed in the triple- negative (HER2-,ER-,PR-) subtype. Both cancers are highly aggressive and resist treatments currently available in the clinic. Because these cancer types often overexpress the ?6?4 integrin as well, we have now examined their dependence on signaling from these receptor complexes. We have discovered that these signaling mechanisms are essential for the growth and survival on the cancer cells, and that their signaling requires the assembly of the integrin and HER2 or EGFR with syndecans, another family of matrix receptors. Indeed, syndecan-1 appears necessary for signaling by HER2??6?4, and syndecan-4 appears to be required by EGFR??6?4. Our goal is to define the molecular details of syndecan assembly with these signaling complexes, develop mutants and blocking peptides that disrupt the organizing function of these two syndecans, and test the mutants and peptides in tumor growth, angiogenesis and the activity of cancer stem cells in animal models of HER2+ and TN breast cancer. The outcome of this work will provide potential insight into the development of new therapeutics to target HER2+ and TN breast cancer.

PROJECT SUMMARY Head & Neck cancer (HNC) is the sixth most prevalent cancer worldwide, with over 600,000 new diagnoses annually. Long-term survival rates for HNC have remained relatively unchanged for the past several decades. Thus, the identification of new targets and therapeutic approaches in HNC are desperately needed. This proposal identifies two new molecular targets in HNC and a new class of inhibitors that show significant promise as new therapies. The molecular targets are signaling complexes organized by syndecans, a family of matrix receptors; they are comprised of integrins, and receptor tyrosine kinases, specifically the epidermal growth factor receptor (EGFR) and insulin-like growth factor-1 receptor (IGF1R). These two kinases are overexpressed in HNC and are known to be causal factors. The integrins that are captured (the ?v?3/?v?5 integrin with IGF1R and the ?6?4 integrin with EGFR) are also known to be causal in HNC, suggesting that the IGF1R- and EGFR-coupled signaling complexes are likely to have central roles in this disease. Competitive peptides (called synstatins or SSTNs) represent a new class of inhibitors that mimic motifs in the extracellular domains of the syndecans and thus block the assembly and signaling of these complexes. Preliminary findings show that the SSTNs block the survival of HNC cells, as well as endothelial cells activated by the tumors to undergo angiogenesis. SSTNs have no effect on normal, resting cells, have no apparent toxicity in animals and are remarkably stable in human plasma and in vivo. We propose to test the hypothesis that these novel synstatins shrink or eradicate HN tumors by targeting the tumor cells as well as the angiogenesis upon which they depend. Specifically, we plan to: (1) Characterize the mechanism of SSTN inhibition of syndecan-coupled EGFR and IGF1R in HNC tumor cells; (2) Evaluate SSTN efficacy in vivo during the progression of HNC from precancerous lesions to tumor formation using the 4-NQO mouse model; and (3) Analyze biomarkers predictive of syndecan-coupled EGFR and IGF1R activity in human tumors and patient-derived HNC xenografts undergoing SSTN therapy. Our goal will be to validate biomarkers that can be used to identify HNC patients that are candidates for SSTN therapy and to demonstrate that SSTNs show significant promise as novel therapeutics for HNC when compared to inhibitors currently in clinical use.

7. PROJECT SUMMARY Multiple myeloma, a disease in which malignant plasma cells reside in the bone marrow, is the second most prevalent hematologic malignancy in the United States. Over 26,000 new cases are diagnosed and over 11,000 die from myeloma each year. Myeloma is a devastating cancer marked by fatigue, intractable bone pain, renal failure and recurrent infections. The emergence of new therapies (e.g., bortezomib, thalidomide) over the past decade has greatly improved survival rates; yet the overall outlook for patients remains grim as these therapies slow rather than cure the disease and patients ultimately relapse, then rapidly decline. Thus, discovering new targets and their inhibitors, especially for relapsed patients, is a high priority. Key interactions within the bone marrow stroma are critical for myeloma cell invasion and survival, including stimulating angiogenesis that supports tumor growth and metastasis. We have recently discovered three novel mechanisms, complemented by three highly promising inhibitors, that govern myeloma cell invasion, survival, the angiogenesis upon which they depend and a novel mechanism of immune suppression by the tumors. All three mechanisms rely on syndecan-1 (Sdc1, CD138), a cell surface heparan sulfate proteoglycan that is highly expressed in myeloma and correlates with poor prognosis in this disease. The three mechanisms involve insulin-like growth factor receptor (IGF1R), which is critical for the survival of myeloma cells, VLA4 (the ?4?1 integrin) that the myeloma cells rely on for extravasation, interactions in the bone marrow necessary for growth and survival and drug resistance, and vascular endothelial growth factor receptor-2 (VEGFR2), which we have recently shown drives the invasion of heparanase-expressing myeloma cells, but also appears to suppress recruitment of NK- and cytotoxic T-cells. In each of these cases, the mechanism is also expressed and active on endothelial cells providing the tumor with a blood supply and metastatic outlets. Each of these important receptors is coupled to the extracellular domain of Sdc1 and their function is blocked by highly stable peptides (synstatins) that we are developing as potential therapeutics for myeloma. We propose in this application to investigate the molecular underpinnings of these mechanisms, and to test our peptides as potential new therapeutics in rigorous mouse models of multiple myeloma.

Head & Neck cancer (HNC) is the sixth most prevalent cancer worldwide, with over 600,000 new diagnoses annually. Epidermal growth factor receptor (EGFR) is overexpressed in HNC and leads to poor prognosis. Surprisingly, however, FDA-approved drugs that inhibit canonical EGFR signaling have had limited success in the clinic, suggesting that disease progression relies on non-canonical mechanisms of EGFR signaling. We have discovered such a non-canonical mechanism that consists of a hexameric receptor complex organized by syndecan-4 (Sdc4) containing EGFR, the hepatocyte growth factor receptor homologue MST1R/RON, the laminin-binding ?3?1 and ?6?4 integrins, and a second syndecan, Sdc2. RON and the cytoplasmic/nuclear kinase c-Abl become constitutively activated when incorporated into this complex, suppressing activation of p38MAPK that would otherwise cause immediate cessation of DNA synthesis and S-phase arrest. A peptide (SSTNEGFR) that represents the extracellular docking site in Sdc4 competitively blocks the formation and signaling of this receptor complex. Preliminary findings show that SSTNEGFR prevents the invasion of HNC cells and induces their rapid cell cycle arrest. Whereas invasion relies on active EGFR, cell cycle progression depends on EGFR but not its kinase activity, identifying a non-canonical EGFR signaling mechanism that is likely to be a critical new therapeutic target in cancers such as HNC that overexpress EGFR. Remarkably, SSTNEGFR does not cause cell cycle arrest in normal oral epithelial cells. Specifically, we plan to: (1) define the molecular organization and signaling mechanism of the Sdc:RTK:ITG complex, focusing on molecular interactions used by the two syndecans to assemble this complex, (2) identify the c-Abl and p38MAPK targets that govern stress signaling and S-phase arrest, and (3) test the efficacy of SSTNEGFR against human HNC patient-derived xenografts (PDXs) and the 4-NQO mouse model of HNC to determine at what stages in the initiation and progression HNC the therapeutic is effective. Our goal will be to understand the molecular underpinnings of this unique receptor complex, understand how it drives HNC and identify it as a possible new target for therapeutics to treat HN disease.