Differential proteomic analysis of a human breast tumor

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Article Differential proteomic analysis of a human breast tumor
and its matched bone metastasis identifies cell membrane
and extracellular proteins associated with bone metastasis
Bruno Dumont, Vincent Castronovo, Olivier Peulen, Noela Bletard, Philippe
Clézardin, Philippe Delvenne, Edwin De Pauw, Andrei Turtoi, and Akeila Bellahcène
J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr201022n • Publication Date (Web): 22 Feb 2012
Downloaded from http://pubs.acs.org on February 29, 2012
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Differential proteomic analysis of a human breast tumor and its matched
bone metastasis identifies cell membrane and extracellular proteins
associated with bone metastasis
Bruno Dumont1, Vincent Castronovo1, Olivier Peulen1, Noëlla Blétard2, Philippe Clézardin3,
Philippe Delvenne2, Edwin A. De Pauw4, Andrei Turtoi*1,4 and Akeila Bellahcène*1
1Metastasis Research Laboratory, University of Liège, Bat. B23, CHU Sart Tilman Liège, B-
4000 Liège, Belgium
2Department of Pathology, University of Liège, Bat. B23, CHU Sart Tilman Liège, B-4000
Liège, Belgium
3INSERM, Research Unit 1033, UFR de Médecine Lyon-Est (domaine Laënnec), F-69372
Lyon Cedex 08, France
4Laboratory of Mass Spectrometry, Department of Chemistry, University of Liège, Bat. B6c,
B-4000 Liège, Belgium
*Authors contributed equally to the study
Corresponding author:
Akeila Bellahcène, Ph.D.
Metastasis Research Laboratory,
GIGA-Cancer, University of Liege,
Tel. [phone]
Fax: [phone]
E-mail: [email protected]
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ABSTRACT
The classical fate of metastasizing breast cancer cells is to seed and form secondary
colonies in bones. The molecules closely associated with these processes are predominantly
present at the cell surface and in the extracellular space, establishing the first contacts with the
target tissue. In this study, we had the rare opportunity to analyze a bone metastatic lesion and
its corresponding breast primary tumor obtained simultaneously from the same patient. Using
mass spectrometry, we undertook a proteomic study on cell surface and extracellular protein-
enriched material. We provide a repertoire of significantly modulated proteins, some with yet
unknown roles in the bone metastatic process as well as proteins notably involved in cancer
cell invasiveness and in bone metabolism. The comparison of these clinical data with those
previously obtained using a human osteotropic breast cancer cell line highlighted an
overlapping group of proteins. Certain differentially expressed proteins are validated in the
present study using immunohistochemistry on a retrospective collection of breast tumors and
matched bone metastases. Our exclusive set of selected proteins supports the set-up of further
investigations on both clinical samples and experimental bone metastasis models that will
help to reveal the finely coordinated expression of proteins that favor the development of
metastases in the bone microenvironment.
Keywords: bone metastasis, breast cancer, primary tumor, matched, proteomics
Abbreviations: BM: bone metastasis; GSSG: oxidized glutathione; PT: primary tumor;
SLRP: small leucine-rich proteoglycan
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INTRODUCTION
Some of the most frequently occurring cancers, including breast, lung, and prostate
adenocarcinomas, predictably metastasize to bone. In the United States, bone metastases
affect more than 400 000 people every year. For advanced breast tumors, 70% of patients will
develop metastasis into the skeleton.1 Although there has been progress in the treatment of
breast cancer, the prognosis of patients is still limited by the occurrence of distant metastases.
Clinically, bone metastases are often associated with high morbidity, including
hypercalcemia, pathological fracture, spinal cord compression, and severe bone pain.
Bisphosphonates are a class of pharmacological agents that inhibit osteoclast-mediated bone
resorption and hence can prevent the morbidity associated with skeletal lesions.2
At the biological level, cancer cells within the tumor mass have to adapt to a particular
microenvironment. For metastases to occur, cancer cells must adjust their phenotype to
survive and grow in their new seeding site. Thus, they must survive in the circulation, seed in
the target organ, extravasate into it, and show persistent growth.3 It is therefore important to
identify the molecular mediators that support the settling of cancer cells in distant organs.4-6
Because it is difficult to obtain suitable clinical specimens, studies on primary tumors
and their matched metastases are rare.7 An obvious reason is that primary breast tumors are
usually surgically removed before the occurrence of metastases. This is particularly true for
breast cancer for which macroscopic metastases may erupt many years after the primary
tumor resection.8 Moreover, the metastases are not removed at all when the disease is well
advanced. In addition, removed metastases are generally small and must be processed intact
for pathology to confirm their metastatic identity, leaving no samples for research.
In breast cancer research, gene expression profiles of matched metastases and primary
tumors from clinical specimens are understudied. Indeed, the few published studies reporting
differential gene expression used primary tumor and associated lymph node metastases
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samples.7 The lack of studies on human metastatic specimens is particularly apparent when it
comes to characterizing the main changes associated with tumor cell dissemination at the
protein level.5, 6 There have been only two original reports describing differential protein
expression on clinical specimens. These proteomic studies compared primary breast tumors
and matched regional lymph node metastases.9, 10 In the context of breast cancer metastasis,
most of the studies were performed with human cancer cell lines having different metastatic
potencies.11, 12 Our group has been involved in several studies aimed at understanding the
molecular mechanisms driving tumor cells to metastasize. Due to the limitations described
above, we have used a transcriptomic approach to identify differentially expressed genes
between MDA-MB-231 human breast cancer cells and their bone metastasis-derived isogenic
clone called B02.13 More recently, we completed this study using a cell membrane proteomic
analysis on the same material.14 So far, no proteomic comparative study on primary breast
cancer and its matched bone metastases has been reported. In the present work, we had the
rare opportunity to collect and investigate a primary breast tumor and its matched bone
metastase collected from a patient at autopsy.
Cell surface and extracellular matrix proteins are the first interface in the complex
dialogue between the tumor and its microenvironment that occurs upon metastatic
dissemination. Proteomic approaches are able to identify and quantify this subset of proteins
with specific cellular localization. However, the extensive dynamic range of the entire cellular
proteome precludes a straightforward proteomic analysis. Therefore, we employed a specific
and comprehensive three-step approach to enrich cell surface and extracellular proteins.15
First, the cell surface proteins were labeled in intact tissue samples with modified biotin and
extracted using affinity chromatography. Second, from the remaining material a selective
enrichment of glycopeptides was performed to identify glycoproteins that are known to be
preferentially secreted or membrane based. The last step involved analysis of the remaining
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