Differential Wnt signalling in bone marrow mesenchymal stem cells from multiple myeloma and MGUS patients
Sarah Essex1, Anandram Seetharam2, 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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