The emerging role of CD44 in regulating skeletal metastasis

Background

Bone metastasis is a frequent and often incurable complication of breast cancer causing severe bone pain, pathological fractures, spinal cord compression and hypercalcaemia. We have focused on establishing the significance of the cell surface hyaluronan receptor CD44 in underpinning the preferential metastasis of breast cancer cells to bone. In prior in vitro studies, we demonstrated that depletion of CD44 expression in breast cancer cells attenuates their adhesion to bone marrow endothelial cells (BMECs). Our recent experiments have also determined that the expression of CD44 is elevated in the bone homing breast cancer subline MDA231BO relative to that detected in the parental MDA231 breast cancer cell line. Together these experiments suggest a physiological role for this receptor in promoting the entry of breast cancer cells into the bone microenvironment.

Methods

To further understand the potential significance of CD44 signalling to breast cancer metastasis, we established a tetracycline-regulated CD44 expression system in the minimally invasive, CD44-negative MCF7F cell line. Removal of tetracycline from the growth media resulted in time-dependent increases in CD44 expression in MCF7F cells, promoting increased cell invasion and migration responses in addition to potentiating the adhesion of MCF7F cells to BMECs. Subsequent microarray analysis was conducted using this expression system to identify CD44/HA regulated genes in breast cancer cells.

Results

The expression and activation of CD44 was associated with increased expression of a subset of genes implicated in metastasis including proteolytic enzymes, growth factors and cytoskeletal proteins (for example, cortactin). Interestingly, the cysteine protease cathepsin K and the matrix metalloprotease MT1MMP were identified as CD44/HA regulated genes. These proteases target collagen I, a major component of the bone matrix whose degradation is a major consequence of osteolytic metastasis of breast cancer. Consistent with their respective metastatic potential, immunoblotting and ELISA based experiments have confirmed that the expression of MT1MMP and cathepsin K are both elevated in the MDA231BO bone homing cells relative to the parental MDA231 cells. In addition, the expression of cathepsin K and MT1MMP in the MDA231BO cells was significantly decreased upon RNAi-mediated suppression of CD44. Quantitative real-time PCR, immunoblotting and ELISA based experiments have also demonstrated that the transcript and protein expression of cathepsin K and MT1MMP increase in response to CD44/HA signalling in a panel of CD44-expressing breast cancer cell lines (MDA231, MDA157 and MCF7F). Currently, we are (i) investigating the mechanistic basis underpinning the transcription of these target genes in breast cancer cells, (ii) determining the functional significance of their overexpression in facilitating breast cancer cells to degrade a collagen I matrix, and (iii) using the MDA231BO cell line to determine the in vivo significance of CD44 expression to osteolytic metastasis.

Conclusion

It is consequently our hypothesis that CD44 may not only promote extravasation into the bone marrow but may also confer an osteoclast-like phenotype to the cancer cell, thus orchestrating the ability of cancer cells to initiate and regulate the modification of the bone matrix. The long-term objective of our research will be to determine whether CD44 expression and that of its transcriptional targets may be predictive for those breast cancer patients at higher risk of developing skeletal disease and/or potentially lead to the development of novel and more effective therapeutic strategies to attenuate bone metastasis.