CD44 signalling increases cathepsin K and MT1MMP expression to potentiate breast cancer cell invasion through collagen I

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 have 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 a bone-homing breast cancer subline MDAMB231BO relative to that detected in the parental MDAMB231 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. Quantitative real-time PCR, immuno-blotting and ELISA-based experiments have 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). Further experiments conducted using a parental and bone-homing subclone of the MDAMB231 cell line (MDAMB213BO) have shown that the expression of CD44, cathepsin K and MT1MMP is elevated in the MDAMB231BO cells relative to their parental counterparts. Furthermore, CD44/HA signalling was shown to increase cathepsin K and MT1MMP mRNA and protein expression in the MDAMB231BO cells. Consistent with their increased metastatic phenotype, MDAMB231BO cells displayed enhanced invasion on HA-supplemented Matrigel and collagen I and demonstrated enhanced collagenolytic activity as demonstrated using an in vitro fluorescence-based assay. RNAi mediated depletion of CD44 and MT1-MMP expression and pharmacological inhibition of cathepsin K attenuated CD44 promoted invasion through a collagen I matrix. We are currently investigating the mechanistic basis underpinning the transcription of these proteases in breast cancer cells, and using the MDA231BO cell line to determine the in vivo significance of CD44 expression to osteolytic metastasis of breast cancer.

Conclusion

Our studies demonstrate that CD44 signalling regulates collagenase activity in breast cancer cells underpinning their invasion through matrix substrates that are enriched within breast tissues and organs to which this disease preferentially metastasises. 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 may potentially lead to the development of novel and more effective therapeutic strategies to attenuate bone metastasis.