Understanding and Targeting the Oncogenic Glycocalyx

Cell surfaces feature abundant glycosylation. Glycans adorn roughly 90% of cell surface proteins and a huge array of phospholipids and sphingolipids comprising the plasma membrane, and myriad polysaccharides bind to cell surface proteins. This collection of glycans concentrated on cell surfaces is known as the glycocalyx and every cell has one. Like many cellular phenomenon, malignant transformation often results in reliable patterns of changes to the glycocalyx. Just as oncogenes are often upregulated in cancers and tumor suppressor genes downregulated, large cell-surface glycoproteins are very often upregulated as well. And tumor cells are often found to be hypersialylated. So consistently upregulated are the mucin class of glycoproteins in cancer, that they are used as biomarkers of the disease. Clearly, investigation into the role of glycans in oncogenesis is warranted./nFor decades, these changes were documented correlatively, but a potential causative role in oncogenesis for these glycans remained elusive. The reasons for our lack of understanding are technical in nature; glycans and glycoproteins are very challenging to study using standard molecular biology techniques. Cancer biologists and cell biologists have been able to use molecular genetics techniques to tease apart the structure-function relationship of most proteins they set their sights on, but glycans are not template encoded. While a protein can be manipulated by changing its corresponding genetic DNA, glycans have no such coding counterpart. Glycosylation patterns are the result of countless metabolic pathways and the balance of activity of glycosyltransferases and glycosidases. So there is no such analogous facile manipulation-to-phenotype technique yet available to glycobiologists./nChemical biology has emerged as the savior to the struggling glycobiologist. By utilizing chemical techniques, glycobiologists now have techniques for gaining traction on some cell-surface glycosylation patterns. Our lab developed just such a technique in the construction of our mucin-mimetic glycopolymers. Through chemical synthesis, we can create glycan-containing molecules that emulate the structure of native glycoproteins. Through a hydrophobic tail, these molecules spontaneously insert into the membranes of cells and thus decorate them with the glycans we chose to incorporate into the molecule. In this way we can begin to ask and answer questions such as: what effect does glycan ‘X’ have upon cancer progression when overexpressed on tumor cell surfaces?/nIn chapter 1, I describe my contribution to these chemical tools. While profoundly useful, the early generation of these mucin-mimetic glycopolymers are constitutively uptaken by the cells which they decorate, meaning there is a limited timespan for the types of experiments they can contribute to. I constructed series of various lipid anchors for the glycopolymers and tested their ability to display the glycopolymers on cell surfaces. Using this empirical approach, we discovered that the synthetic sterol cholesterylamine is capable of promoting the recycling of our glycopolymers after endocytosis and thus display them for days to weeks. We go on to show that these mimics of mucin structure are capable of endowing cancer cells with metastatic-like phenotypes in a zebrafish model of metastasis./nIn chapter 2 we put these molecules to use and answer the question: what contribution to metastasis do mucin glycoproteins make on tumor cell surfaces? Mucins are overexpressed on a massive number of cancers. It is estimated that 65% of all tumors diagnosed each year in the US overexpress MUC1—just one member of this family of cell-surface glycoproteins, of which there are at least twenty! They are found to positively correlate with metastasis, but the mechanism for such a contribution has remained entirely elusive. We found—using our mucin-mimetic glycopolymers and confirmed with genetic MUC1 constructs—that the mucin ectodomain has a unique contribution to the adhesion of cancer cells in the metastatic niche. When a cell arrives at a distal site in order to form a metastatic lesion, it must not only survive there but also proliferate. The cell may very well find that there isn’t much adhesive support for such behavior. In our models, overexpressing mucin ectodomains allows it to grow and survive despite that shortcoming. This work not only provides the first mechanistic support of a role for the mucin ectodomain in metastatic spread, but it has dramatic consequences for the targeting of mucin oncogenes. Work has been done to target the biochemically active cytoplasmic tails of mucins with novel therapeutics. Our studies imply that such targeting is futile and may not combat the mucin’s most deadly structural contribution to oncogenesis: its ectodomain./nSo how, then, are biomedical scientists to produce drugs which might combat this dramatic contribution of the cancer cell glycocalyx to progression of the disease? In chapter 3, I describe a radically new approach to the treatment of cancer—but also to medical therapies in general. Using a handful of chemical biology techniques, we synthesized a conjugate of a monoclonal antibody and an enzyme. The enzyme can exert its effect on the cell surface once brought into proximity by the antibody. In this way, enzymes that modify the glycocalyx in an anti-cancer manner can be directly targeted to tumors. As a proof of principle, we first made sialidase-trastuzumab conjugates. High expression of sialic acids has long been associated with cancer, but it was only recently that their role in immune-evasion has been appreciated. By displaying a lot of sialic acid on their cell surfaces, tumors are able to bind to inhibitory receptors on immune cells— which might otherwise kill the cancer cells—and avoid their destructive powers. Upon removing those sugars, we have seen that they become susceptible to the immune cells once again. Therefore, in this chapter I demonstrate how an enzyme that cleaves sialic acids can be selectively targeted to tumor cells bearing the antigen of the antibody, and their killing by immune cells enhanced as much as 2 fold, via this new class of therapeutics: the antibody-enzyme conjugates.

Advisor: Bertozzi, Carolyn R