A View Into Cell Trafficking Through A Rolling Receptor.  A membrane protein displaying key carbohydrate moieties is characteristically expressed by circulating T cells or tumor cells engineered to enter distinct tissues (such as skin and bone as shown).  These carbohydrate moieties interact with specific counter-receptors found on the luminal aspect of the microvasculature wall, resulting in target cell rolling and targeted tissue tropisms.  

The following research Projects in The Dimitroff Laboratory are funded by grants from The American Cancer Society and National Institutes of Health.  These research studies are designed to elucidate how prostate cancer cells metastasize through prostate cancer cell surface glycoconjugates and how glycoconjugate-modifying agents, such as 4-fluoro-glucosamine, which has been shown to dampen inflammation, modulate the immune system.

Project 1:  Role of PSGL-1 in the Bone Metastasis of Prostate Cancer
Prostate cancer (PCa) metastasis characteristically occurs in bone and often results in the poor prognosis of patients.  Unfortunately, there are no treatments that specifically target the molecular pathogenesis of bone metastasis.  Recent studies have indicated that PCa cells exhibit an enhanced adhesion for bone blood vessels compared with adhesion to blood vessel linings in other tissues.  The Dimitroff Laboratory has shown that initial adhesive interactions between bone-metastatic PCa cells and bone blood vessels are mediated by PSGL-1.  Remarkably, expression analysis of PSGL-1 on tissue micro-arrays (TMA) of normal prostate epithelium and of localized and metastatic PCa tissue has revealed that PSGL-1 is almost exclusively found on metastatic PCa cells with the highest level expressed on PCa cells in bone.  The objective of studies outlined in this project is to elucidate the molecular pathogenesis of PCa metastasis to bone and determine whether PSGL-1 promotes bone-metastatic behavior of PCa and whether PSGL-1 possesses structural motif(s) for targeting of novel anti-cancer therapies.  The specific aims are: 1.) To analyze the glyco-biochemistry of PSGL-1 on metastatic PCa cells and 2.) To validate the functional role of PSGL-1 in PCa metastasis, in vivo.  Experimentation includes innovative glycobiology, biochemistry and cell adhesion technologies to ascertain the critical structures conferring the “rolling” activity of PSGL-1 on metastatic PCa cells.  In vivo analysis is being performed using mice transplanted with human bone to explore the efficiency of PSGL-1⁺ or PSGL-1^- human PCa cells to home (traffic) into human bone (Please see Figure 1 depicting the transplantation of human bone tissue onto mice and the fidelity of human bone vessel markers).  Biochemical perturbation of PSGL-1 structural motifs critical for functional rolling activity with rationally-designed agents is also being performed in this in vivo investigation to reveal potential therapeutic strategies for pharmacologic development.  Results from these analyses will offer new insights into the molecular pathogenesis of prostate tumor metastasis and may provide rationale for development of new therapies targeting homing receptors.

Figure1.  (A) Human Bone Xenotransplantation.  Fresh trabeculae human bone is resected from the diaphysis a femoral head with a 4mm dermal punch scalpel and transplanted subcutaneously on the dorsa of immunodeficient mice.  After a 4-week engraftment period, a viable, highly vascularized human bone xenograft is evident in the mouse.  (B) Detection of Human Blood Vessel Receptors in a Human Bone Xenograft.  The expression of human bone blood vessel marker (stained in brown) demarcates functional human blood vessels in transplanted human bone tissue. PSGL-1 counter-receptors (stained in brown) are found on human bone blood vessels in human bone xenografts.

Project 2:  Analysis of the Glycobiological Control of Prostate Cancer Metastasis

Human prostate cancer (PCa) characteristically metastasizes to bone, and yet there is little known about the major factors that instigate this pathologic phenomenon in patients with advanced PCa.  It is clear that PCa cell motility and invasiveness are malignant phenotypes that are important for the formation of bone metastases.  However, preferential adhesion of metastatic PCa cells to human bone marrow (BM) microvessels suggests that molecular interactions between PCa cells and BM endothelial cells (BMEC) augment metastatic potential and confer the specificity of PCa metastasis to bone.  

Considering the molecular mechanism for mediating hematopoietic stem cells (HSC) trafficking to bone, The Dimitroff Laboratory hypothesizes that bone-homing receptors (or E-selectin ligands (ESL)) commonly found on HSC are also expressed and functional on circulating PCa cells and help confer the capacity of PCa cells to enter bone.  Since BMEC constitutively express endothelial (E)-selectin, which binds ESLs on HSC and helps direct HSC trafficking to bone, Dr. Dimitroff believes that this molecular receptor – ligand pair also directs PCa cells to bone.  The Dimitroff Laboratory has recently published data showing that ESLs, namely PSGL-1, on PCa cells do indeed initiate adhesive contact with BMEC.  Regarding the regulation of ESL synthesis, it is well-known that ESL function on HSC is conferred by carbohydrate moieties, represented principally by sialyl Lewis X (sLe^X), that are synthesized by α1,3 fucosyltransferases (FTs).  FTs are well-established regulators of E-selectin-binding and trafficking of HSC, but investigations on the role of FTs in PCa cell – BMEC binding and metastasis have not been undertaken.  Dr. Dimitroff hypothesizes that FTs control the function of ESL on PCa cells and enhance the metastatic potential of PCa.  (Please see Figure 2, which illustrates this hypothetical model.) 

Current and future research in this component of The Dimitroff Research Program will be designed to analyze the expression of FTs in authentic localized and metastatic PCa tissue and to perform mechanistic analysis to determine whether FTs are involved in sLeX synthesis on PCa cells to confer E-selectin-binding activity and elicit bone-metastatic activity.  The molecular identity and structural binding determinants created by FTs will provide rationale for the development anti-metastatic therapeutics against these new targets.  If the enzymatic requirements for ESL synthesis in human PCa cells are identical to those needed for ESL on leukocytes, Dr. Dimitroff believes that the use of non-toxic glycoconjugate modifiers, such fluorinated analogs of natural-occurring sugars (i.e. 4-F-GlcNAc) also developed by The Dimitroff Laboratory, will allow for an innovative treatment intervention during early stages of disease progression.  The promise of this potential anti-metastatic therapeutic can only be appreciated from knowledge of molecules identified in these studies. 

Figure 2.  Hypothetical Model of Circulating PCa Cell Migration to Bone and the Role of α1,3 Fucosyltransferases (FT3, 6 and 7) in the Production of PCa Cell E-selectin Ligands

Project 3:  Analysis of Immune Modulation by Analogs of Glucosamine 

Trafficking of leukocytes to skin is directed by adhesive interactions between vascular endothelial selectins and leukocyte selectin ligands.  Leukocyte selectin ligand activity is conferred by specialized carbohydrate structures displayed on leukocyte surface proteins.  These specialized carbohydrates expressed on distinct subsets of leukocytes impart the capacity of leukocytes to enter skin and are, thus, otherwise referred to as skin-homing receptors.  To this end, controlling the migration of skin-homing leukocytes associated with skin disorders, such as atopic dermatitis, allergic dermatitis, psoriasis and cutaneous leukemias, with selectin ligand-modifying sugar mimetics represents a potentially promising therapeutic strategy.  The effects of over-the-counter glucosamine formulations have been purported to relieve arthritic conditions and are supportive of this notion.  Preliminary data from The Dimitroff laboratory show that a simple fluorinated glucosamine analog (or fluoro-glucosamine) of naturally-occurring glucosamine modulates carbohydrate structures required for leukocyte selectin ligand activity, causing attenuated cutaneous inflammatory responses.  (Please see Figure 3, which illustrates how fluoro-glucosamine interferes with the elongation of carbohydrate chain formation). The Dimitroff laboratory hypothesizes that this fluoro-glucosamine analog can be utilized as a model of Complementary and Alternative Medicinal (CAM) agents, such as glucosamine, to study how and which glycans on effector leukocytes are sensitive to glyco-metabolic inhibition.  The objectives of studies in this project are: 1.) To define the mechanism of fluoro-glucosamine action on leukocyte selectin ligand and other adhesion molecule expression and 2.) To investigate the in vivo efficacy and specificity of fluoro-glucosamine treatment in mouse models of inflammation.  The Dimitroff Laboratory aims to improve the understanding of how fluoro-glucosamine inhibits dermatotropic activity of effector leukocytes and to determine whether fluoro-glucosamine affects leukocyte adhesion molecule function involved in other non-skin migration patterns.  Numerous biochemical approaches using fresh and cultured leukocytes are being employed to study how fluoro-glucosamines modify leukocyte glycan expression and function.  Fluoro-glucosamine efficacy and specificity are also being analyzed on effector skin- and non-skin-homing leukocyte subsets using well-defined models of inflammation.  The overall goal of this preclinical investigation is to show how fluoro-glucosamines behave as immunomodulators of cutaneous inflammation, providing insight into potential CAM glucosamine efficacy on leukocyte homing receptors.

Figure 3.  (A) Biosynthesis of Sialyl Lewis X-containing Core 2 O-linked Carbohydrates or Selectin Ligands in the Presence of Glyco-metabolic Inhibitor, Peracetylated-4-Fluorinated-D-glucosamine (4-F-GlcNAc) (Panel B).