Lessons from Experimental Hybrids
I first encountered the fusion theory of metastasis in the early 1990s and was struck by its similarity to a well-known feature of evolutionary genetics—fusion of different cell types as a mechanism for creating new gene-expression patterns and biodiversity.
With this large biological precedent as a stimulus, my research group created experimental hybrids in the laboratory between normal macrophages ultimatelyand a mouse melanoma cell line that was only weakly metastatic in mice. We were amazed to find that many of the hybrid clones were now markedly metastatic when implanted into mice.
This finding led us to a now 15-year study of the fusion theory. We have studied experimental macrophage-melanoma hybrids from many angles—chromosome content, genes expressed that are associated with metastasis, capability for chemotactic migration, and many other features. We were looking for similarities of hybrids to metastatic melanoma in humans. The thought was that the extent to which experimental macrophage-melanoma hybrids might resemble metastatic melanoma cells in humans could provide support and direction for the theory, though not ultimate proof.
We established a long list of molecules expressed by macrophage-melanoma hybrids that were also characteristic of metastatic melanoma cells and normal macrophages alike. Thus this “quacks like a duck” approach gave us some confidence that we might be on the right track—we could create a phenotype similar to that of authentic metastatic melanoma cells by fusing normal macrophages with non-metastatic melanoma cells.
But, not only did the experimental hybrids express a number of metastasis-associated proteins, they also expressed macrophage-like glycosylation structures associated with migration and metastasis. This involved a leukocytic glycosylation system where complex sugar structures known as β1,6-branched oligosaccharides are attached to N-glycoproteins, thereby orienting the cell toward a migratory phenotype.
A rate-limiting enzyme in this system is N-acetylglucosaminyltransferase V (GnT-V), a Golgi complex enzyme that is highly expressed in macrophages and other migratory leucocytes and also widely associated with metastasis.
GnT-V and its enzymatic products, b1,6-branched oligosaccharides conjugated to N-glycoproteins, are associated with poor outcome in melanoma and carcinomas of the breast, colon, lung, GI tract, and endometrium.
GnT-V is a promiscuous enzyme with many N-glycoprotein substrates such as motility-associated integrins, adhesion molecules, and growth factor receptors. As such, GnT-V is a master regulator of migration and metastasis, initiating cascade reactions by activating numerous protein substrates through altered glycosylation. Our results with macrophage-melanoma hybrids could explain why so many cancers express GnT-V and β1,6-branched oligosaccharides while their normal cell counterparts do not.