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Anna
M. Masellis: Dr. Masellis received her B.Sc. degree from the University
of British Columbia in Vancouver, British Columbia and her Ph.D. degree
from the University of Alberta in Edmonton, Alberta. Her Ph.D. work
focused on the role of the tetraspan molecule CD9 and integrin adhesion
molecules in B cell adhesion. She also studied B cell interaction with
bone marrow stromal cells, which directed her interest to postdoctoral
studies in adhesion and migration of bone marrow derived plasma cells
in multiple myeloma. She joined the Virginia Piper Cancer Institute
at Abbott Northwestern Hospital in Minneapolis in 1998 and is an adjunct
Assistant Professor in the Department of Medicine at the University
of Minnesota. Her current research focus is tumor cell interactions
with bone and includes hyaluronan-cell interactions in mesenchymal cell
differentiation and disease.
Introduction
Tumorigenesis is a multistage process, having an absolute requirement on coordinated events intrinsic as well as external to the cancer cell. It is well recognized that the tumor microenvironment plays a significant role in tumor progression. In particular, soluble as well as extracellular factors, including cytokines and growth factors, extracellular matrix molecules and cellular ligands, all influence the biological behavior of the tumor. Hyaluronan, a major constituent of the extracellular matrix, is a large negatively charged glycosaminoglycan composed of repeating units of N-acetylglucosamine and glucuronic acid. Hyaluronan has important roles in matrix assembly, cell proliferation, cell migration and embryonic/tissue development. Recent studies have provided evidence for hyaluronan and hyaluronan-cell interactions affecting not only cell migration and proliferation, but also the invasive potential of cells and malignant transformation. The biological functions of hyaluronan may be in part attributed to its physical properties (being a large negatively charged macromolecule), its associations with other extracellular matrix molecules (including aggrecan, versican, neurocan, brevican, TSG-6, link proteins) and involvement in signal transduction through interaction with cell surface receptors (CD44, Receptor for Hyaluronan Mediated Motility, Lyve-1).
The
Bone Marrow Microenvironment
The bone marrow microenvironment is a multi-functional network of cells
and extracellular matrix that maintains hematopoietic cell proliferation,
differentiation and survival (Fig. 1) 1.
Among the important cell lineages from the bone marrow are those derived
from the mesenchymal progenitor cell, including chondrocytes, adipocytes,
fibroblasts and osteocytes 2.
These cells differentiate along the various lineages in response to external
microenvironment signals and internal lineage commitment signals. However
these external and internal signals are not well understood. It is likely
that many factors, including growth factors, cytokines and cellular as
well as extracellular matrix, impact growth, differentiation and function
of the bone marrow mesenchymal cells.
 |
| Fig. 1. The Bone Marrow in Hematopoiesis |
The bone marrow extracellular matrix consists
primarily of fibronectin, collagen types I and IV, laminin and the glycosaminoglycans
heparan sulfate, chondroitin sulfate and hyaluronan. Hyaluronan, a major
glycosaminoglycan of this matrix functions in multiple roles to influence
bone marrow hematopoiesis and cellular differentiation. The influence
of CD44:hyaluronan interaction in hematopoiesis has been demonstrated
by the use of anti-CD44 mAbs or treatment with hyaluronidase; both treatments
significantly reduce lymphopoiesis and myelopoiesis in vitro
3, 4.
Our laboratory is interested in how the expression of hyaluronan and hyaluronan
synthase (HAS) enzymes in bone marrow mesenchymal cells, as well as how
hyaluronan-mediated cellular interactions, might impact on the localization
and survival of multiple myeloma plasma cells and other bone seeking tumors.
Over the past several years, we have utilized an in vitro model
system of bone marrow stromal cultures derived from both healthy donors
and patients with multiple myeloma to define a role for hyaluronan in
malignancies with bone involvement.
Hyaluronan and Tumorigenesis
Hyaluronan and hyaluronan synthases appear to have a significant biological
role in tumor progression, and, in certain malignancies, hyaluronan serves
as a clinical prognostic indicator of disease progression. There is increasing
evidence that hyaluronan and hyaluronan synthase genes are intricately
involved in tumorigenesis, and in the process of angiogenesis 5-7.
In particular, hyaluronan production is up-regulated in a variety of malignant
tissues compared to normal cells, and increased hyaluronan levels as a
consequence of HAS gene expression have been shown to correlate with increased
tumor cell invasion 8-14.
For example, over-expression of Has2 in fibrosarcoma cells or of Has3
in prostate carcinoma cells results in growth of larger tumors, while
breast cancer cells transfected with Has1 have increased metastatic potential
compared to parental cells. While the majority of studies have utilized
solid tumor models, there is evidence that hyaluronan is relevant in hematological
malignancies as well. Most recently in multiple myeloma, hyaluronan levels
in serum are shown to correlate with poor disease outcome 15.
Multiple Myeloma
Multiple myeloma is an incurable plasma cell malignancy that accounts
for 13% of lymphoid malignancies in the Caucasian population 16.
This disease is characterized by the presence of monoclonal bone marrow
plasma cells within the bone marrow (hereafter referred to as myeloma
plasma cells), monoclonal protein, hypercalcemia, lytic bony lesions and
anemia. An illustration of multiple myeloma development during B-cell
maturity is shown in Figure 2. Treatment
for myeloma is not curative, and the median survival is 3-4 years.
The bone marrow microenvironment likely plays a profound role in myeloma
disease progression. The fact that myeloma plasma cells preferentially
accumulate in the bone marrow implies that the bone marrow mesenchymal
cells within this compartment best support the growth and maintenance
of the myeloma clone. Co-culture systems have shown that physical cell-cell
contact, soluble and matrix-associated growth factors/interleukins, and
cell-matrix interactions affect the growth and survival of the myeloma
bone marrow plasma cell and its response to chemotherapeutic agents (refer
to Fig. 1). In addition to myeloma bone marrow
plasma cells, there is speculation that a CD19+ B cell (hereafter
referred to as myeloma B cell) exists within the peripheral blood of myeloma
patients that may comprise a component of the malignant clone with the
ability to intravasate from the peripheral blood into the bone marrow.
Others and we have shown that bone marrow mesenchymal cells derived from
myeloma patients have altered adhesion receptor expression, cytokine expression
and extracellular matrix expression. Despite the increasing acceptance
of the influential role imparted by the bone marrow microenvironment on
myeloma cells, few investigations have focused on the characterization
of the bone marrow stroma, and the identification of any specific anomalies
within this compartment, that may contribute to the pathophysiology of
this disease.
 |
| Fig. 2. B Cell Development and Multiple Myeloma |
Hyaluronan Synthase in Normal and Multiple Myeloma Bone Marrow Cells
The overall metabolism of hyaluronan is determined by the enzymatic activities
of hyaluronan synthases and hyaluronidases. Three hyaluronan synthase
genes have been described, each encoding a plasma membrane protein responsible
for hyaluronan synthesis 17.
Although expression of any one hyaluronan synthase gene is sufficient
for hyaluronan synthesis, the contribution of the three HAS gene products
to hyaluronan production and expression in most systems is not yet clear.
It has been suggested that the hyaluronan synthases possess unique enzymatic
properties resulting in different kinetics of hyaluronan synthesis. For
example, it is suggested that Has3 is responsible for the synthesis of
low-molecular mass hyaluronan (< 2 x 105) while Has1 and
Has2 are responsible for the synthesis of larger molecular weight hyaluronan
(>2 x 106). Likewise, potential differences in enzymatic
function of the various hyaluronan synthases may be very important in
vivo and possibly relate to the various functions ascribed to hyaluronan.
As a first step towards discerning the role of hyaluronan as an integral
component of the bone marrow extracellular matrix in multiple myeloma
patients, the hyaluronan synthase gene expression and hyaluronan production
in bone marrow mesenchymal cells derived from healthy donors and from
multiple myeloma patients were characterized. The expression of hyaluronan
synthase genes in healthy and myeloma bone marrow mesenchymal cells was
determined by quantitative competitive Rt-PCR. Utilizing this method,
it has been shown that bone marrow mesenchymal cells derived from myeloma
patients express predominantly Has1. In contrast, bone marrow mesenchymal
cells derived from healthy donors predominantly express Has2 (Fig.
3).
 |
| Fig. 3. cRt-PCR
for Has1 and Has2 in Myeloma and Healthy Donor Bone Marrow Mesenchymal
Cells Total RNA was extracted from bone marrow mesenchymal cell cultures at no greater than 80% confluency. cRt-PCR was performed as indicated in reference 18. The cRt-PCR was performed with 1  g/ml
total RNA and recombinant Has DNA used as the internal competitor
(constructed as described in ref. 18).
The concentration of the Has competitor ranged from 10 pg/L
to 0.000 0001 pg/L
(a range of 0.001-10 pg/l
is shown). |
 |
| Fig. 4. A Schematic of Hyaluronan Synthase, Hyaluronan Receptors and Hyaluronan-Responsive Cytokines in the Myeloma Bone Marrow Microenvironment |
| Table. 1. Relative Amounts of Hyaluronan in Medium and Cell Layers of Bone Marrow Mesenchymal Cells |
 |
| Fluorophore-assisted carbohydrate electrophoretic (FACE) analysis was used to measure the relative amounts of hyaluronan in cultures derived from either healthy donors or myeloma patients. Medium and cell layer fractions of 48-hour cultured cells were subjected to proteinase K digestion, ethanol precipitation and digestion with hyaluronidase SD, chondroitinase ABC and glucoamylase. To visualize carbohydrates, sample were subjected to fluorescent-derivatization with 2-aminoacridone and electrophoresed using MONOP composition gels. Data were obtained in collaboration with Dr. A. Calabro (Department of Biomedical Engineering, Cleveland Clinic Foundation). See reference 18 for further materials and methods. |
The role of Hyaluronan in Multiple Myeloma
Recently, both abnormally low or high hyaluronan concentrations in serum
have been shown to correlate with disease progression in multiple myeloma
15. Although there
is yet no indication that abnormal hyaluronan levels induce shorter overall
survival of patients, it may be that hyaluronan serum level may be indicative
of the pathological course of this disease. In that regard, hyaluronan
levels in the serum may be prognostic for a select group of myeloma patients
with adverse outcome. Although the clinical usefulness of hyaluronan in
myeloma is not yet certain, it is apparent from a multitude of studies
(cited below) that hyaluronan and hyaluronan receptors are involved in
a variety of biological aspects in this disease.
Hyaluronan Receptors and Adhesion in Myeloma:
Myeloma cells express multiple adhesion receptors, including the hyaluronan
receptors CD44 and RHAMM (Receptor for Hyaluronan Mediated Motility) 19.
With regards to CD44, myeloma plasma cells exclusively express CD44v3,
CD44v6 and CD44v9 (20) while
normal bone marrow plasma cells do not express these isoforms. Clinically,
the expression of isoform CD44v9 by myeloma plasma cells is correlated
with adverse prognosis. In keeping with the unique expression of CD44
isoforms, Crainie et al. have shown that myeloma plasma cells express
unique isoforms of RHAMM not found in healthy donor B cells 21.
In addition to a full length isoform of RHAMM, myeloma plasma cells express
RHAMM-48, which has a deletion of 48 bp, and RHAMM-147, which has a deletion
of 147 bp. Presently however, the role of these isoforms in hyaluronan-binding
is unknown. Interestingly, RHAMM is also expressed on a subset of myeloma
bone marrow fibroblasts. The expression of RHAMM is confined to the intracellular
compartment of cultured bone marrow mesenchymal stromal cells (Fig.
5), with no cell surface membrane expression detected by either
fluorescence microscopy or flow cytometry. As with RHAMM expression in
myeloma plasma cells, the expression of intracellular RHAMM is distinct
to myeloma bone marrow fibroblasts and is not found in healthy donor fibroblasts
nor bone marrow fibroblasts derived from patients with chronic lymphocytic
leukemia, lymphoma, acute myeloid leukemia or hairy cell leukemia (Fig.
5). At this time, the function of intracellular RHAMM in hyaluronan
binding, or the particular isoform of RHAMM expressed by myeloma mesenchymal
stromal cells is not yet defined.
 |
| Fig. 5. Expression
of RHAMM in Myeloma Bone Marrow Mesenchymal Cells Myeloma-derived bone marrow mesenchymal stromal cells were grown to sub-confluency in  -MEM
supplemented with 10% FBS and culture confluency determined by light
microscopy (10x objective) [A]. Monolayers were permeabilized
in 70% ethanol at 4
and fixed in 1% formalin prior to staining for distribution of RHAMM
using the anti-RHAMM mAb 3T3.5,
followed by incubation with anti-mouse Ig conjugated to phycoerythrin.
Stained cells were visualized and immunofluorescent images captured
by laser confocal microscopy (40x objective). A subset (20%) of myeloma
mesenchymal stromal cells expressed intracellular RHAMM as shown in
[B], although CD44 was undetectable on these same
cells (not shown).
Images 4A and 4B were not derived from identical cultures. |
 |
| Fig. 6. Hyaluronan
in Myeloma Plasma Cell Adhesion to Bone Marrow Mesenchymal Stromal
Cells Bone marrow mesenchymal stromal cells were derived from newly diagnosed, untreated myeloma patients or healthy donor bone marrow aspirates. Cultures were initiated by plating 1 x 106 cells/ml in -MEM supplemented
with 10% FBS.
In some experiments, the mesenchymal stromal cell monolayers were
pre-treated with 0.1 U/ml chondroitinase ABC (filled bars) or 40 TBU
Streptomyces hyaluronidase (hatched bars), for 2 hrs, prior
to addition of myeloma plasma cells labeled with 5 M
BCECF-AM (Molecular Probes, Eugene OR). Although chondroitinase ABC
can also degrade hyaluronan, treatment of monolayers with chondroitinase
ABC showed only 5% inhibition while treatment of monolayers with Streptomyces
hyaluronidase resulted in 40-50% inhibition of adhesion. |
 |
| Fig. 7a. Binding
of Hyaluronan to Myeloma Plasma Cells Plasma cells were analyzed for their ability to bind to hyaluronan-FITC (100 g/ml) (B),
in the presence or absence of 10 g/ml
(C) anti-CD44 (50B4) or (D) anti-RHAMM
mAb (3T3.5). Avidin-FITC (100 g/ml)
and a non-specific IgG1 isotype control murine mAb (10 g/ml)
were used as controls (A and E respectively).
|
 |
| Fig. 7b. Transmigration
Response of Myeloma Cells in response to Hyaluronan and Fibronectin To examine the transmigration of myeloma plasma cells, a modified Boyden chamber assay was used. 1 x 105 plasma cells were placed in the upper chamber of a transwell insert containing either 100 g/ml
hyaluronan or 20 g/ml
fibronectin and examined for their ability to migrate to the underside
of filters after 1.5 hours of culture. Migrated cells were counted
under inverted microscopy (magnification 20x). Results are expressed
as the average of cells per field (minimum of 10 fields counted).
|
mg/ml),
protection from apoptosis and an increase in cell proliferation was observed.
This hyaluronan-mediated survival and proliferation effect was associated
with the down-regulation of p27kip1 expression and the hyperphosphorylation
of Rb, two molecules involved in cell-cycle progression through G1.
Interestingly, the survival and proliferation effects of hyaluronan on myeloma
plasma cell lines did not require CD44:hyaluronan interaction, but did involve
signaling through the gp-130 IL-6 receptor subunit, suggesting the possibility
of an hyaluronan-initiated autocrine feedback loop for IL-6 production and
utilization.Our group has demonstrated that hyaluronan stimulation of the plasma cell line ARH77, with hyaluronan concentrations that promote myeloma cell migration, results in the activation of Raf-1 and MAP kinases (Table 2). The activation of Raf-1 kinase was rapid and reproducible, with phosphorylation of MAP kinase kinase-GST occurring as early as 3 minutes and declining to base levels by 15 minutes. The increase in Map kinase activity ranged between 2-10 fold (Table 2). Furthermore, hyaluronan stimulation resulted in the up-regulation of IL-1
mRNA, which was inhibitable by the MEK inhibitor PD98059 (Fig.
8) indicating that IL-1
up-regulation occurs through a signal transduction pathway involving MEK.
The production of IL-1 by
myeloma plasma cells is of significance to the pathophysiology of the disease.
IL-1 is a potent osteoclast
activating factor and plays a role in the development of lytic bone lesions,
a common and devastating clinical feature in multiple myeloma. IL-1
also up-regulates cytokine production, including IL-6 which is a major cytokine
crucial to myeloma cell growth. Thus, it could be speculated that in myeloma
patients, hyaluronan expressed by the bone marrow mesenchymal cells may
initiate a chain of events, as a consequence of interactions with hyaluronan
receptors on the plasma cells, that may lead to up-regulation of cytokines
involved in the manifestation of bone disease as well as in myeloma plasma
cell growth.| Table. 2. Hyaluronan Stimulation Activates Raf-1 and MAP kinase in Myeloma Plasma Cells |
 |
| The myeloma cell line ARH77 was serum
starved for a minimum of 4 hours prior to stimulation with 100 g/ml
hyaluronan and assay for kinase activity. In some experiments, cells
were pre-treated with the MEK inhibitor PD98059 for 30 minutes prior
to stimulation, and the inhibitor was maintained in the medium during
hyaluronan stimulation. Cells were activated with hyaluronan at concentrations
ranging from 10 – 100 g/ml
and maximal Raf-1 kinase was observed at 100 g/ml.
Thus, this concentration of hyaluronan was utilized in all subsequent
experiments. Cell lysates were prepared; Raf-1 or MAP kinase immunoprecipitated,
and in vitro kinase assays carried out using MEK-GST or MBP
as substrates, respectively. In all experiments, fold increase was
determined relative to the activity of each kinase at time 0. nd=
not done |
 |
Fig. 8. Rt-PCR
for IL-1 and
IL-6 in Myeloma Plasma Cells after Hyaluronan Stimulation Rt-PCR for IL-1
and IL-6 was performed on the myeloma ARH77 cells stimulated with
either hyaluronan [HA] (100 g/ml)
or fibronectin [FN] (20 g/ml)
for 1, 6 and 12 hours. Unstimulated ARH77 cells express neither IL-1
nor IL-6. Rt-PCR products were run on a 1.5% agarose gel and stained
with ethidium bromide. Shown is a representative of 3 independent
experiments. The arrow highlights the IL-1
Rt-PCR product stimulated by HA at 6 hours and by FN at 1 hour. The
stimulation of IL-1
message by hyaluronan is completely inhibited by the MEK inhibitor
PD98059. Rt-PCR product for IL-6 was undetectable. |
Concluding Remarks
While the management of multiple myeloma and other hematological malignancies
has focused on the eradication of the malignant cell, it is important
to consider the impact of the marrow microenvironment on tumor response
and normal cell function. In addition to the presence of malignant plasma
cells, our data suggest that the bone marrow microenvironment in myeloma
is abnormal possibly due to its exposure to tumor cells and/or chemotherapeutic
agents. In particular, in multiple myeloma, hyaluronan synthesis may have
prognostic value for a subgroup of patients. in vitro studies
with myeloma plasma cells implicate hyaluronan in many functions associated
with tumorigenesis, including elevated expression of hyaluronan receptors,
enhanced cell migration and cell adhesion and altered cell signaling.
Thus, the management of myeloma will depend not only on eliminating tumor
cells but also on the re-establishment of the bone marrow microenvironment
from one that supports tumor growth to one that supports normal cellular
function. Increased insights into the role of hyaluronan in myeloma may
eventually lead to the use of hyaluronan as a novel therapeutic target
in myeloma. For instance, hyaluronan oligosaccharides may function to
block migration, to block adhesion of plasma cells in the bone marrow,
or to inhibit the establishment of cytokine loops established between
the bone marrow mesenchymal cell and the myeloma plasma cell as a consequence
of cell-cell interaction. Likewise, hyaluronan could serve a greater role
as a prognostic tool. In that regard, the presence of different molecular
weight hyaluronan, possibly as determined by HAS enzyme expression, could
be used to identify patients with poor outcome. Based on the cited literature
and data from our laboratory, we propose that in myeloma, hyaluronan synthesis
and matrix organization in the bone marrow imparts a unique function to
the bone marrow environment that may impact not only myeloma plasma cell
growth and survival but also impact on normal bone marrow mesenchymal
cell function.
West DC, Kumar S: Hyaluronan and angiogenesis. Ciba Found Symp., 143, 187-201, 1989
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