Differential regulation of the sphere formation and maintenance of cancer-initiating cells of malignant mesothelioma via CD44 and ALK4 signaling pathways

Malignant mesothelioma (MM) has a poor prognosis and is largely resistant to standard treatments, so it is important to seek novel therapeutic strategies for this disease. Cancer-initiating cells (CICs) were previously identified in MM using stem cell-associated markers in combination with spheroid cultures. However, the mechanisms underlying the induction and maintenance of CICs in MM remain to be fully explored. Here we showed that the CICs, which had high aldehyde dehydrogenase levels (ALDHbright) and stem cell-associated genes, were expanded in MM cells cultured under sphere-forming conditions. The MM spheroids also initiated tumors in immunodeficient mice more efficiently than did conventional adherent MM cells. In the MM spheroids, the expression of hyaluronan (HA) synthases was upregulated. Inhibiting the HA synthesis or CD44 functions by gene knockdown or neutralizing antibody abolished the formation of large-sized spheroids and the expansion of ALDHbright CICs. The expression of activin-A was also increased in the spheroids, and type I activin-A receptor subunit (ALK4) was upregulated in the ALDHbright CICs. The neutralization of activin-A or functional inactivation of ALK4 diminished the ALDHbright CICs without affecting spheroid formation. The knockdown of CD44 or ALK4 strongly suppressed the tumor growth in immunodeficient mice. These results together suggest that the HA–CD44 and activin-A–ALK4 pathways differentially regulate the spheroid formation and maintenance of ALDHbright CICs in MM cells, and that both pathways play critical roles in tumor growth in immunodeficient hosts. Our findings provide a novel therapeutic option for MM that targets signaling pathways that promote the CIC compartment through CD44 and ALK4.