Haematologica 2003 - Supplements

6. Role of microenvironment
6.1 Cell adhesion
136
Regulation of SDF-1-CXCR4 interactions by SOCS3 in
Myeloma and MGUS bone marrow.
Natalia Gonzalez-Paz, Michelle Manske, Tammy L. Price-
Troska, Michael Timm, Gregory Ahmann, Rafael Fonseca,
Roshini S. Abraham.
Division of Hematology, Hematology Research Program,
Department of Internal Medicine, Mayo Clinic, Rochester, MN
Multiple myeloma (MM), among the hematological malignancies
is second only to lymphoma in incidence and mortality statistics.
From published data, it is evident that interactions between the
bone marrow microenvironment and myeloma cell play an
important role in the pathogenesis of disease. Our data show that
the stromal cell-derived chemokine, SDF-1, transcripts are
significantly upregulated in the bone marrow of myeloma (p =
0.04) and MGUS (p = 0.04) patients compared to normal
controls. SDF-1 protein is also increased in the bone marrow
plasma of myeloma and MGUS patients. Overexpression of
SDF-1 and its interactions with its receptor, CXCR4, has been
implicated in preventing apoptosis and stimulating growth of
tumor cells in a number of neoplastic diseases, through activation
of Erk1/2, PI3-K/Akt and members of the JAK-STAT family.
Myeloma and MGUS plasma cells from patients as well as
several myeloma cell lines tested showed expression of CXCR4.
Short exposure to SDF-1 induces internalization of the CXCR4
receptor through the GRK2/arrestin pathway. Jurkat, a T cell line
with constitutive expression of CXCR4 (positive control) showed
internalization of more than half of the CXCR4 receptor
molecules on the cell surface after exposure to 50ng/ml SDF-1
for 30 minutes. The IL-6-dependent myeloma cell line, KAS-6/1
also showed a similar reduction in CXCR4 expression while DP-
6 showed only about 20% reduction. This may indicate
dysregulation of the GRK2 pathway in some myeloma cell lines.
Prolonged exposure to SDF-1 functionally inactivates CXCR4,
without altering surface expression, through the suppressor of
cytokine signaling –3 protein (SOCS3) and is mediated by the
JAK-STAT pathway. We hypothesize that the difference
between the ‘benign-pre-myeloma’ state of MGUS and myeloma
lies not so much in the plasma cell but in the bone marrow
microenvironment and its regulation through SOCS3 and
possibly other proteins. SOCS3 transcripts are expressed
constitutively in bone marrow stromal cells (BMSCs) cultured
from patients with myeloma, MGUS or normals. SOCS3 mRNA
is also seen in the IL-6-dependent myeloma cell lines, ANBL-6,
KAS-6/1 and DP-6. SDF-1 transcripts are present in BMSCs
from normals, MM and MGUS as well as in the 3 IL-6 MM cell
lines. Interestingly, very preliminary observations indicate that
the SOCS3 protein, as detected by Western blot, is seen only in
BMSCs derived from MGUS and normal bone marrow but not in
MM BM. We are in the process of validating and verifying this
finding. However, if this is true, it would suggest that while
levels of SDF-1 are comparable in MM and MGUS BM, the
interaction of SDF-1-CXCR4 is differentially modulated by
SOCS3. In other words, in the MGUS BM, the interaction of
SDF-1 with CXCR4 is appropriately controlled by SOCS3, which
prevents continual activation of the receptor. However, the
absence of SOCS3 regulation in the MM BM would allow SDF-
1-CXCR4 interactions to go unchecked, leaving the downstream
signaling pathways constantly activated, triggering proliferation
and block of apoptosis of the transformed plasma cell. Thus,
SOCS3 may play a crucial role in determining the fate of the
clonal plasma cell through SDF-1-CXCR4 interactions.
137
Myeloma cell transendothelial migration and invasion
in response to chemokine stromal cell-derived factor-1
Marisa Parmo-Cabañas, Natalia Wright, Andrés Hidalgo,
Angelika M. Drager and Joaquin Teixidó
Centro de Investigaciones Biológicas, Department of Immunology,
Madrid, Spain, and Department of Hematology, Vrije Universiteit
Medical Center, Amsterdam, The Netherlands.
Multiple myeloma is a B cell neoplasm characterized by
accumulation of myeloma cells in the bone marrow (BM) in
contact with stromal cells. Myeloma cells express the integrin
α4ß1 which mediates their attachment to VCAM-1 and
fibronectin displayed on BM stroma. Using purified recombinant
forms of these α4ß1 ligands we previously reported that SDF-1α
a chemokine present in the BM microenvironment, modulated
α4ß1-dependent myeloma cell adhesion. In the present work we
show that SDF-1triggers transendothelial migration of MM-
CD38hiCD45RA- BM cells and the myeloma-derived cell line
NCI-H929, involving a transient upregulation of α4ß1-dependent
myeloma cell adhesion. In addition, a key role for the small GTPase
RhoA in the increase of this adhesion by SDF-1α is suggested
by the inhibition exerted by C3 exozyme, which interferes with
RhoA activation. We also report here that SDF-1promotes
myeloma cell in vitro invasion across Matrigel, which is mediated
by the metalloproteinase MT1-MMP, whereas MMP-9 played
minor roles. These data indicate that SDF-1α/CXCR4 axis in MM
might contribute to myeloma cell transendothelial migration and
lodgement in the BM microenvironment involving modulation of
α4ß1 adhesive activity. Additionally, MT1-MMP and MMP-9
activities expressed by myeloma cells could mediate their
invasion across basement membranes after their transendothelial
migration in response to SDF-1α
138
Endoplasmic reticulum stress induces apoptosis in
myeloma cells
Miki Nakamura, Hiroyuki Hata, Takashi Sonoki,Shima
Uneda, Yutaka Okuno, Hiro Tatetsu, Masataka Mori,
Tomomi Gotoh and Hiroaki Mitsuya.
Department of Internal Medicine II, Kuamomoto University School
of Medicine
Introduction: The endoplasmic reticulum (ER) is the primary
organelle in which proteins are synthesized and modified for
appropriate folding. Accumulation of unfolded proteins in the ER
lumen, which is caused by various environmental stresses, leads
to unfolded protein response (UPR) which results in activation of
XBP–1(X–box binding protein–1), a transcriptional factor
required for the expression of ER chaperons. When stresses are
present in excess, cells undergo apoptosis through the caspase–12
pathway. Multiple myeloma (MM) cells bear abundant ER,
prompting us to ask whether MM cells are under ER stress and
whether ER stress in MM cells can be controlled.
Materials and Methods: Plasma cells from MM patients were purified
using negative selection utilizing magnetic beads. The cDNA coding
XBP-1 (a marker for ER stress) was obtained through RT-PCR of
mRNA purified from MM cells of 34 cases and subjected to
digestion with Apa–LI. Apoptosis was detected either
S148