Haematologica 2003 - Supplements

118
IL-6- Induced Phosphorylation of Gab-Family Proteins
in MM Cells is Src- family tyrosine kinase dependent
Podar-K1, Mostoslavsky-G2, Tai-YT1, Sattler-M1, Catley-
LP1, Hideshima-T1, Chauhan-D1, Mulligan-RC2,
Anderson-KC1
1Dana-Farber Cancer Institute, Department of Medical Oncology,
Harvard Medical; 2School Children’s Hospital, Department of
Genetics, Harvard Medical School
Interleukin-6 is a potent growth and survival factor in multiple
myeloma (MM), which initiates signaling pathways via binding
to the interleukin-6 receptor. Early IL-6 mediating and
modulating signaling events, which ultimately lead to the
activation of extracellular signal-regulated kinase (ERK) and
phosphatidylinositol 3-kinase, are poorly understood. Our studies
show for the first time that Gab-family adaptor proteins Gab1 and
Gab2 are expressed by multiple myeloma cells and that IL-6
mediates their phosphorylation and association with several
signaling proteins. In addition, we demonstrate that IL-6- induced
phophorylation of both Gab1 and Gab2 and their association with
several downstream signaling proteins are Src-family tyrosine
kinase- dependent. Consequently, inhibition of Src-family
tyrosine kinase by PP2 significantly reduced the activation of the
downstream signaling molecules ERK and Akt-1, leading to
significant reduction of MM cell proliferation and survival. These
studies identify Src-family tyrosine kinases and downstream
adaptor proteins as potential new therapeutic targets in multiple
myeloma.
119
Activation of the Erk5 route by IL-6 in multiple myeloma
Xonia Carvajal-Vergara, Soraya Tabera, Azucena Esparís-
Ogando, Norma Gutiérrez, Gemma Mateo, Jesús San
Miguel, Atanasio Pandiella.
Centro de Investigación del Cáncer, Instituto de Micobiología
Bioquímica, and Hospital Universitario de Salamanca
Several signalling pathways have been implicated in the
transduction of proliferation/survival responses. An important
group of pathways is represented by the MAPK routes. Three
classical MAPK pathways have been described in mammals: the
extracellular signal-regulated kinase 1 and 2 (Erk1 and Erk2), the
p38 and the Jun N-terminal kinases (JNK) routes. A novel MAPK
pathway, the Big MAPK-1/Erk5 pathway, has recently been
implicated in proliferative responses. The role of Erk5 in MM
biology is still unknown. Studies on the expression of Erk5 in
different MM cell lines indicated that the protein was expressed
in all the cell lines investigated. The subcellular distribution of
Erk5 was analysed by immunofluorescence microscopy, and in
the different cell lines studied the antibody stained the cytoplasm
of the cells. Some Erk5 staining was also localized in the nuclear
compartment. Staining was prevented by preincubation with the
peptide antigen against which the antibody had been raised,
indicating that staining was specific. To analyze whether IL-6, a
major survival and proliferation factor for MM cells, regulated
the Erk5 pathway we treated MM cells with this factor and
analysed Erk5 activation by Western blotting. Addition of 10 nM
IL-6 to MM1S, MM1R and MM144 cells caused Erk5 to migrate
as a more retarded band. The action of IL-6 on Erk5 activation
was found to be time- and dose-dependent. Maximal effect on
Erk5 activation was observed around 20 minutes of treatment
with IL-6, and 5 nM of IL-6. At present we are evaluating the
importance of the Erk5 route on MM proliferation by the use of a
form of Erk5 mutated in the TEY microdomain.
120
GENE EXPRESSION PROFILING AS A MEANS TO
UNDERSTAND MYELOMA CELL GROWTH CONTROL:
IDENTIFICATION OF MEK-DEPENDENT IL-6 AND IGF-I
INDUCED GENES
B.K. Arendt*, R.C. Tschumper*, K.V. Ballman+, G.A.
Stolovitzkya, and D.F. Jelinek*
Depts. of Immunology* and Biostatistics+, Mayo Clinic, Rochester,
MN 55905; and IBM TJ Watson Research Centera, Yorktown
Heights, NY 10598
Multiple myeloma (MM) is an invariably fatal disease that
accounts for approximately 1-2% of all human cancers. Although
progress has been made in better understanding growth control of
MM cells, further investigation is required. We have been
interested in using a genomic profiling approach to facilitate an
understanding of the mechanism by which interleukin 6 (IL-6), a
known growth factor for MM cells, stimulates tumor cell growth.
We, and others, have also shown that insulin-like growth factor I
(IGF-I) can also directly stimulate myeloma cell growth,
particularly when IL-6 is present. The ability of IL-6 and IGF-I
to stimulate myeloma cell growth is of interest because the
receptors for these two growth factors are strikingly different.
Thus, the IL-6 receptor requires non-receptor tyrosine kinases,
e.g., Jaks, to initiate signaling downstream of gp130, whereas the
IGF-I receptor is a receptor which itself has tyrosine kinase
activity. Of interest, the proliferative responses stimulated by
both IL-6 and IGF-I are substantially inhibited when cells are
cultured in the presence of the highly specific MEK (MAPKK)
inhibitor, U0126. Our goal in this study was to use gene
expression profiling to identify genes induced by these two
growth factors alone or in combination, and to use the U0126
MEK inhibitor as a tool to specifically focus on genes
downstream of the Raf-MEK-ERK pathway.
Three MM cell lines were cultured with and without IL-6 and
IGF-I and the MEK inhibitor for 24 hours before measuring DNA
synthesis and isolating total RNA. IL-6 stimulation induced 17-,
46-, and 2-fold increases in DNA synthesis in the DP-6, KAS-
6/1, and KP-6 cell lines, respectively and IGF-I stimulation
resulted in 3-, 16-, and 4.5-fold increases, respectively. In each
of the three cell lines, U0126 inhibited IL-6, IGF-I, and IL-
6+IGF-I stimulated responses by an average of 80%. For gene
expression analysis, cRNA was prepared from each sample and
hybridized to Affymetrix HG-U95Av2 gene biochips.
Expression data were analyzed using recently developed
algorithms that combine data normalization with a holistic-model
analysis of the probe data. We have also employed permutation
test methods to calibrate significance values. Finally, to better
understand the interrelationships of groups of genes, we have also
applied the pattern recognition software, Genes@Work. Of
interest, we have identified a significant number of genes that are
induced in both a MEK-dependent and MEK-independent
manner and these results will be presented. Notable examples of
MEK-dependent IL-6 induced genes included several currently
unknown genes, bFGF, flotillin-1, HSP-70, c-fos, CTPsynthetase,
hnRNP, CSE-1, Ran/Tc4, LAS-1, and the Cdc45-like
PORC-PL gene. Many of these genes have been associated with
other human cancers and play roles in cell cycle control.
However, our studies also link many of these genes for the first
time with the Raf-MEK-ERK pathway. Because of the role that
the Raf-MEK-ERK pathway plays in myeloma cell proliferation,
this approach has the potential to specifically identify genetic
targets important in tumor cell growth.
S140