Deacetylated Sialic Acid Sensitizes Lung and Colon Cancers to Novel Cucurbitacin-Inspired Estrone Epidermal Growth Factor Receptor (EGFR) Inhibitor Analogs.

Cancers utilize sugar residues such as sialic acids (Sia) to improve their ability to survive. Sia presents a variety of functional group alterations, including O-acetylation on the C6 hydroxylated tail. Previously, sialylation has been reported to suppress EGFR activation and increase cancer cell sensitivity to Tyrosine Kinase Inhibitors (TKIs). In this study, we report on the effect of deacetylated Sia on the activity of three novel EGFR-targeting Cucurbitacin-inspired estrone analogs (CIEAs), MMA 294, MMA 321, and MMA 320, in lung and colon cancer cells. Acetylation was modulated by the removal of Sialate O-Acetyltransferase, also known as CAS1 Domain-containing protein (CASD1) gene via CRISPR-Cas9 gene editing. Using a variety of cell-based approaches including MTT cell viability assay, flow cytometry, immunofluorescence assay and in-cell ELISA we observed that deacetylated Sia-expressing knockout cells (1.24-6.49 µM) were highly sensitive to all CIEAs compared with the control cells (8.82-20.97 µM). Apoptosis and varied stage cell cycle arrest (G0/G1 and G2/M) were elucidated as mechanistic modes of action of the CIEAs. Further studies implicated overexpression of CIEAs' cognate protein target, phosphorylated EGFR, in the chemosensitivity of the deacetylated Sia-expressing knockout cells. This observation correlated with significantly decreased levels of key downstream proteins (phosphorylated ERK and mTOR) of the EGFR pathway in knockout cells compared with controls when treated with CIEAs. Collectively, our findings indicate that Sia deacetylation renders lung and colon cancer cells susceptible to EGFR therapeutics and provide insights for future therapeutic interventions.

Deacetylated sialic acids modulates immune mediated cytotoxicity via the sialic acid-Siglec pathway.

Cancers utilize glycans to evade the immune system via the Sialic acid (Sia)-Siglec (Sialic-acid-binding immunoglobulin-like lectins) pathway. Specifically, atypical structural forms of sialic acid bind to inhibitory Siglec receptors on natural killer (NK) cells resulting in the suppression of immune cell mediated cytotoxicity. The mechanism of action that governs the Sia-Siglec pathway in cancers is not understood. Specifically, how deviations from the typical form of Sia mechanistically contribute. Here, we focused on modulating 9-O and 7, 9-O-acetylation of Neu5Ac, via CRISPR-Cas9 gene editing, a functional group that is absent from Sias on many types of cancer cells. The two genes that are responsible for regulating the level of acetylation on Neu5Ac, are Sialic acid acetylesterase (SIAE) and Sialic acid acetyltransferase (CASD1). These genes modulated Siglec binding in colon, lung and a noncancerous kidney cell line. In the absence of SIAE, Neu5Ac is acetylated, engagement of cancer associated Siglecs is reduced while binding was increased when the ability to acetylate was removed via CASD1 knock out. In the absence of SIAE NK mediated cytotoxicity increased in both colon and lung cancer cells. In addition to modulating Siglec binding, SIAE expression modulates the level of Sias in a cell, and the α2-6-linkage of Sias-which is specifically upregulated and associated with cancers. Uncovering how functional group alterations on Neu5Ac contribute mechanistically to both Siglec receptor binding, the Sia-Siglec immune evasion pathway, and the production of cancer associated glycosidic linkages-offers a promising avenue for targeted cancer immune therapies in the future.