Illegitimate WNT signaling promotes proliferation of multiple myeloma cells.

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Illegitimate WNT signaling promotes proliferation
of multiple myeloma cells.
Patrick W. B. Derksen, Esther Tjin, Helen P.
Meijer, Melanie D. Klok, Harold D. Mac
Gillavry, Marinus H. J. van Oers, Henk M.
Lokhorst, Andries C. Bloem, Hans Clevers, Roel
Nusse, Ronald van der Neut, Marcel Spaargaren,
and Steven T. Pals, Proceedings of the
National Academy of Science (PNAS) April 20 , 2004
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What is Multiple Myeloma?

• Multiple myeloma (also known as myeloma or plasma
  cell myeloma) is a progressive hematologic
  (blood) disease.
• It is a cancer of the plasma cell, an important
  part of the immune system that produces
  immunoglobulins, or antibodies, to help fight
  infection and disease.
• Multiple myeloma is characterized by excessive
  numbers of tumor cells that expand in the bone
  marrow leading to pancytopenia, an abnormal
  reduction in the number of RBCs, WBCs and blood
  platelets in the blood.
• Overproduction of intact monoclonal
  immunoglobulin (IgG, IgA, IgD, or IgE) or
  Bence-Jones protein (free monoclonal k and L
  light chains) also occurs.
• Transformation of a normal B cell into a
  malignant plasma cell involves a multi-step
  process that includes multiple genetic
  abnormalities.

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• Most of the evolution takes place in the bone
  marrow indicating that signals from the bone
  marrow microenvironment, which may include
  paracrine growth factors, play a critical role in
  sustaining the growth and survival of MM cells
  during tumor progression.

• Resulting plasma cells become malignant, meaning
  they continue to divide unchecked, generating
  more plasma cells that are malignant.
• These myeloma cells travel through the
  bloodstream and collect in the bone marrow, where
  they damage tissue.
• Hypercalcemia, anemia, renal damage, increased
  susceptibility to bacterial infection, and
  impaired production of normal immunoglobulin are
  common clinical manifestations of multiple
  myeloma.

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• It is often also characterized by diffuse
  osteoporosis, usually in the pelvis, spine, ribs,
  and skull.
• Multiple myeloma is the second most prevalent
  blood cancer after non-Hodgkin's lymphoma. It
  represents approximately 1 of all cancers and 2
  of all cancer deaths.
• The median age at diagnosis is about 71 years,
  and only 2 of cases are diagnosed in individuals
  under the age of 45.
• Approximately 50,000 Americans currently have
  myeloma, and the American Cancer Society
  estimates that approximately 15,270 new cases of
  myeloma will be diagnosed during 2004.
• Although a tremendous amount of work has gone
  into the search for the cause of multiple
  myeloma, to date no cause for this disease has
  been identified.

Information found at www.multiplemyeloma.org
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Authors Questions
Do myeloma cells overexpress b-catenin and
nonphosphorylated b-catenin? Do b-catenin and
nonphosphorylated b-catenin get overexpressed in
the primary myelomas in the bone marrow? Can
Multiple Myeloma cells respond to WNT
signaling? Does WNT signaling affect the
localization of b-catenin? Does WNT signaling
control the proliferation of myeloma cells? Does
the disruption of b-catenin/TCF activity effect
Multiple Myeloma proliferation? Is the WNT
pathway in MM cells intact? Is there a
regulatory component?
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WNT signaling

• WNT signals are one class of paracrine growth
  factors that could act to influence the growth of
  MM cells
• WNT signal transduction components, in particular
  adenomatous polyposis coli (APC), and b-catenin,
  are often mutated in cancers and sustained
  overexpression of WNT genes can cause cancer.
• WNT proteins themselves are able to promote the
  proliferation of progenitor or stem cells
• The main event in the WNT signaling pathway is
  the stabilization of b-catenin
• Signaling by WNT factors blocks GSK3b activity,
  resulting in the accumulation of
  nonphosphorylated b-catenin, which will
  translocate to the nucleus

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• Once in the nucleus non phosphorylated b-catenin
  joins T cell factor (TCF) transcription factors
  to drive transcription of target genes
• It has been shown that uncontrolled b-catenin/
  TCF activity plays a big role in many human
  cancers
• The involvement of the WNT pathway in the
  regulation of the survival and expansion of
  progenitor and stem cells, in combination with
  its oncogenic potential in nonlymphoid cells,
  made the authors want to test whether
  deregulation of the WNT pathway occurs in
  lymphoid neoplasia.

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Overview of WNT pathway

• Without WNT signaling, b-catenin exists in its
  phosphorylated form which is tagged with
  ubiquitin and destroyed.
• With WNT signaling unphosphorylated b-catenin is
  able to enter the nucleus, bind to TCF, displace
  groucho and drive transcription of several genes
  including c-myc.

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Do myeloma cells overexpress b-catenin and
non-phosphorylated b-catenin?

• Figure 1A shows that all myeloma cell lines
  tested contained significant b-catenin levels and
  most of them contained detectable levels of
  non-phosphorylated b-catenin.

• Figure 1B compares normal B-cells and plasma
  cells to MM cells.
• This assay shows that there is a big difference
  between the expression of b-catenin and
  nonphosphorylated b-catenin in MM cells vs.
  normal B-cells and plasma cells.

Yes, myeloma cells do overexpress both forms of
b-catenin
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Do b-catenin and non-phosphorylated b-catenin get
overexpressed in primary myelomas in the bone
marrow?

• Figure 1C demonstrates that b-catenin is
  expressed in 9 out of 10 primary MM bone marrow
  samples
• Non-phosphorylated b-catenin is also detectable
  in most primary MM bone marrow samples

• Figure 1D was used to show that 7 out of 7 normal
  bone marrow samples do not express b-catenin or
  non-phosphorylated b-catenin

Yes, both forms of b-catenin are overexpressed in
primary myelomas
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Can MM cells respond to WNT signaling?

• Stimulation with LiCl resulted in increased
  b-catenin and non-phosphorylated b-catenin

• Stimulation with Wnt3a (a specific Wnt protein)
  produced similar results

Yes, myeloma cells do respond to Wnt signaling
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Fig. 2b LiCl induces accumulation of b-catenin
and increased nuclear localization of
nonphosphorylated b-catenin.
Does WNT Signaling affect the localization of
b-catenin?

• Apart from causing b-catenin accumulation, WNT
  signaling also affected the localization of
  b-catenin.
• Before LiCl stimulation, low amounts of b-catenin
  were detected in the cytoplasm and nucleus of MM
  cells by confocal laser scan microscopy.

b-catenin
Non-phos b-catenin
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Does WNT Signaling affect the localization of
b-catenin?

• Stimulation with LiCl led to an increase in the
  total amount of b-catenin (located to the plasma
  membrane at cell-cell contact sites,) as well as
  in the nucleus
• Most of the nonphosphorylated b-catenin was
  localized in the nucleus.

Yes, WNT signaling affects the localization of
b-catenin
Fig. 2b LiCl induces accumulation of b-catenin
and increased nuclear localization of
nonphosphorylated b-catenin.
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Figure 2c. Expression and nuclear localization of
b-catenin in primary MMs.
Does WNT Signaling affect the localization of
b-catenin?
Control
Nuclear staining of non-phos b-catenin (blue)
Isotype-matched antibody in combo with the
anti-Ig light-chain antibodies

• Nonphosphorylated b-catenin was present in the
  nucleus of primary myelomas.
• Nuclear localization of nonphosphorylated
  b-catenin was also detected in primary myelomas,
  but the levels varied among individual cells
  within a given tumor.

Yes, WNT signaling affects the localization of
b-catenin
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Fig 3a. Exogenous Wnt3a promotes proliferation
of MM cells.
Does WNT signaling control the proliferation of
myeloma cells?

• Cell lines were cultured in the presence of L
  cell-conditioned medium (white bars) or
  conditioned medium derived from Wnt3a-transfected
  L cells (black bars)
• 3H thymidine incorporation was measured after
  1, 2, and 3 days of culture.

MM cell lines

• Myeloma cells responded to stimulation with
  Wnt3a-conditioned medium with a 2- to 4-fold
  increase in proliferation and was readily
  observed within the first 24 hours.

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Figure 3b. Purified Wnt3a promotes proliferation
of MM cells
Does WNT signaling control the proliferation of
myeloma cells?

• Cells were cultured in the absence or presence of
  purified Wnt3a in serum-free medium.
• 3H thymidine incorporation was measured after 2
  days of culture.

MM cell lines

• Similar results were also obtained with purified
  Wnt3a, demonstrating the specificity of the
  effect of Wnt3a and ruling out indirect effects
  of other growth factors released as a result of
  autocrine stimulation in the Wnt3a-transfected L
  cells.

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Fig. 3c. LiCl stimulation promotes proliferation
of MM cells.
Does WNT signaling control the proliferation of
myeloma cells?

• Cells were cultured in the absence or presence of
  2mM LiCl in serum-free medium.
• 3H thymidine incorporation was measured after 3
  days of culture.

MM cell lines

• Treatment with LiCl (which inhibited GSK3b
  resulting in b-catenin accumulation Fig 2a.) gave
  rise to a similar increase in proliferation.

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Fig. 3d b-catenin S33Y promotes proliferation of
MM cells.
Does WNT signaling control the proliferation of
myeloma cells?

• Cells were transfected with empty vector or with
  b-catenin S33Y
• 3H thymidine incorporation was measured after 2
  days of culture.

Yes, WNT signaling controls the proliferation of
Myeloma cells
MM cell lines

• Enhanced proliferation was also observed after
  expression of the b-catenin S33Y mutant.

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Fig. 4a DTCF4 inhibits proliferation of MM cells
Does the disruption of b-catenin/TCF activity
effect Multiple Myeloma proliferation?

• MM cell lines OPM1 and NCI H929 were transfected
  with either DTCF or empty vector, both in combo
  with pEGFP constructs
• After overnight culture, viable GFP-positive
  cells were sorted and 3H thymidine
  incorporation was measured after 2 and 3 days of
  culture.

Yes, disruption of b-catenin/TCF activity causes
inhibition of MM proliferation
MM cell lines

• Transfection with DTCF4 (a dominant negative form
  of TCF) strongly inhibited proliferation of the
  MM cell lines OPM1 and NCI H929, which both
  contain large amounts of active b-catenin.

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This suggests.

• WNT signaling is constitutively active, but not
  maximally activated and sensitive to regulation.
  
• So to corroborate these conclusions..

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Fig. 4b TCF reporter activity in MM cells.

• OPM1 cells were either transfected with
  luciferase reporter constructs (pTOPFLASH), alone
  or in combo with b-catenin S33Y and DTCF4 and
  were assayed for luciferase activity.

• OPM1 showed a moderate constitutive b-catenin/TCF
  activity.

• This activity was inhibited by contransfection
  of DTCF4

• A strong receptor activity was obtained after
  cotransfection of the active b-catenin
  mutant S33Y.

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Fig. 4c Wnt3a stimulates TCF reported activity in
MM cells.

• OPM1 and NCI H929 cells were transfected with
  pTOPFLASH, alone or in combo with b-catenin S33Y.
• The cells were either stimulated or not
  stimulated with purified Wnt3a, and were assayed
  for luciferase activity.

• TCF reporter activity was increased by
  stimulating MM cells with purified Wnt3a

Yes, the WNT pathway in MM cells are
constitutively active
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• Is there a regulatory component?
• (the presence of an autocrine activation loop?)

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Table 1. WNT Expression in myelomas and normal B
lineage cells

• To explore this possibility, the authors
  assessed the expression of WNT genes previously
  demonstrated to be expressed within the
  hematopoietic environment.

• Neither normal B cells nor plasma cells expressed
  these WNTs

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Table 1. Continued.

• Expression of WNT5a and WNt10b were found in all
  myeloma cell lines tested, but only WNT16
  transcripts were found in one MM cell line.

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Table 1. Continued.

• In highly purified MM cells, they detected
  expression of WNT5a and/or WNT10b and, in one
  case, WNT16

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Table 1. Continued.

• WNT5a and WNT10b expression was also found in
  bone marrow stromal cells suggesting they may
  function as a paracrine source of WNTs within
  the bone marrow microenvironment

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Finding Answers
1. Do myeloma cells overexpress b-catenin and non
phosphorylated b-catenin? Yes (see Fig 1A B)
2. Do b-catenin and nonphosphorylated b-catenin
get overexpressed in primary myelomas in the
bone marrow? Yes (see Fig 1C D)
3. Can MM cells respond to Wnt Signaling? Yes
(see Fig 2A)
4. Does Wnt Signaling affect the localization of
b-catenin? Yes (see Fig 2B C)
5. Does Wnt signaling control the proliferation
of myeloma cells? Yes (see Fig 3A-D)
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Finding Answers Cont

• 6. Does the disruption of b-catenin/TCF activity
  effect Multiple Myeloma proliferation?
• Yes (Figure 4a.)
• 7. Is the WNT pathway in MM cells intact?
• Yes (Figures 4b 4c.)
• 8. Is there a regulatory component?
• Yes, WNt5a and WNt10b may function as a
  paracrine source of WNTs within the bone marrow
  microenvironment

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Conclusions

• Since normal plasma cells are fully
  differentiated, the activation of the signaling
  routes that causes cell proliferation is
  important for their transformation into MM cells.
• -this study showed WNT signaling can
  control the proliferation of MM
• This means the WNT pathway may prove to be a good
  target for MM therapy.
• More research is needed to determine which WNT
  target genes might be involved in WNT induced
  proliferation in MM