The
malignancy of a
tumor depends on the capability of
cancer cells to metastasize. The process of
metastasis involves cell invasion through connective tissue and transmigration through endothelial monolayers. The expression of the
glycosylphosphatidylinositol-anchored receptor CD24 is increased in several
tumor types and is consistently associated with increased
metastasis formation in patients. Furthermore, the localization of β1-integrins in
lipid rafts depends on CD24. Cell invasion is a fundamental biomechanical process and usually requires cell adhesion to the extracellular matrix (ECM) mainly through β1 heterodimeric
integrin receptors. Here, we studied the invasion of A125 human
lung cancer cells with different CD24 expression levels in three-dimensional ECMs. We hypothesized that CD24 expression increases
cancer cell invasion through increased contractile forces. To analyze this, A125 cells (CD24 negative) were stably transfected with CD24 and sorted for high and low CD24 expression. The invasiveness of the CD24(high) and CD24(low) transfectants was determined in three-dimensional ECMs. The percentage of invasive cells and their invasion depth was increased in CD24(high) cells compared with CD24(low) cells. Knockdown of CD24 and of the β1-integrin subunit in CD24(high) cells decreased their invasiveness, indicating that the increased invasiveness is CD24- and β1-integrin subunit-dependent. Fourier transform
traction microscopy revealed that the CD24(high) cells generated 5-fold higher contractile forces compared with CD24(low) cells. To analyze whether contractile forces are essential for CD24-facilitated cell invasion, we performed invasion assays in the presence of
myosin light chain kinase inhibitor
ML-7 as well as
Rho kinase inhibitor
Y27632. Cell invasiveness was reduced after addition of
ML-7 and
Y27632 in CD24(high) cells but not in CD24(neg) cells. Moreover, after addition of
lysophosphatidic acid or
calyculin A, an increase in pre-stress in CD24(neg) cells was observed, which enhanced cellular invasiveness. In addition, inhibition of the
Src kinase or STAT3 strongly reduced the invasiveness of CD24(high) cells, slightly reduced that of CD24(low) cells, and did not alter the invasiveness of CD24(neg) cells. Taken together, these results suggest that CD24 enhances cell invasion through increased generation or transmission of contractile forces.