Differential Wnt signalling in bone marrow mesenchymal stem cells from
multiple myeloma and MGUS patients
Sarah Essex1, Anandram Seetharam 2, Angelique Barkhuizen2, Wenbin Wei1, Supratik Basu2, Andrew
Filer3, Guy Pratt4, Paul Moss1,5
1School
of Cancer Sciences, University of Birmingham, Birmingham, 2Department of Haematology,
The Royal Wolverhampton Hospitals NHS Trust, Wolverhampton, 3School of Immunology and
Infection MRC Centre for Immune Regulation, University of Birmingham, Birmingham 4Centre for
Clinical Haematology, University Hospital Birmingham NHS Foundation Trust, 5Department of
Haematology, Heart of England NHS Foundation Trust, Birmingham.
Multiple myeloma (MM) is a malignant disease of plasma cells within the bone marrow and has a
premalignant stage termed monoclonal gammopathy of undetermined significance (MGUS) which
progresses to MM at a rate of 1% per year.
Both primary and malignant plasma cells have an absolute requirement for localisation within the
bone marrow niche. Moreover, co-culture of tumour cells and stroma taken from patient bone marrow
at different stages of disease demonstrates that it is the stroma, rather than the tumour cell, that acts
as a major determinant of disease progression. The nature of the critical interactions between the
tumour cell and its microenvironment has yet to be determined in detail but are they are likely to offer
novel therapeutic potential.
We have investigated the role of the bone marrow mesenchymal stem cells (BMMSC) in the
progression of MM. Firstly by isolating and growing BMMSC from control, MGUS and MM patient
bone marrow samples. Then by looking at the full genetic profile of these cells using microarrays, with
detailed pathway analysis to determine the genes involved in disease progression. Next, further
exploring these key genes using RT-PCR, methylation status and splice variant analysis.
30 patients BMMSC were analysed using microarrays; this highlighted 74 genes that had over a 2 fold
difference in expression between control and disease BMMSC. Pathway analysis of these genes
generated several pathways differentially expressed between patients, with wnt signalling being the
most evident. Two wnt pathway genes which are significantly decreased in disease BMMSC,
compared to control, are secreted frizzled-related proteins (sFRPs) 2 and 4. This decrease in
expression was then confirmed by RT-PCR with a concurrent increase in methylation status
suggesting gene silencing. Splice variant analysis of these particular genes showed a differential
expression of exons, which may be functionally significant for wnt signalling.
sFRPs have been shown to be downregulated in many cancers, including breast and colorectal
cancer. The increase in methylation of the sFRP genes in this study silences their expression. The
decrease in sFRP production increases canonical wnt signalling, thus activating target genes, such as
those involved in cell proliferation and differentiation. Conversely, it is known that osteogenic
differentiation of BMMSC into osteoblasts is markedly impaired in multiple myeloma. This is thought to
be due to myeloma-stromal cell contact and/or the release of soluble factors as interleukin-7,
hepatocyte growth factor, interleukin-3, and Wnt inhibitors such as DKK-1. Our results, in keeping with
Giuliani et al (2007), do not support an inhibition of canonical Wnt signalling in the bone marrow
microenvironment. For the first time we show profound switching off of negative regulators of wnt
signalling within MM BMMSC and also importantly this is seen at the MGUS stage. Further work will
help understand these abnormalities of Wnt signalling within BMMSC in MM and MGUS.
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