01.10 ST6Gal-1 Mediates Resistance via Apoptosis and Increases After Treatment in Rectal Cancer Organoids

M. G. Smithson1, R. Irwin1, G. Williams1, M. C. McLeod1, S. Bellis2, K. Hardiman1  1University Of Alabama at Birmingham, Surgery, Birmingham, Alabama, USA 2University Of Alabama at Birmingham, Cell, Development, And Integrative Biology, Birmingham, Alabama, USA

Introduction: Rectal cancer affects more than 44,000 patients annually. Traditionally, rectal cancer is treated with chemotherapy, radiation, followed by surgery but response to this treatment is variable. ST6Gal-1 is an enzyme that adds sialic acid (SA) to cell surface proteins and can modify function. Our previous studies have shown that ST6Gal-1 mediates resistance in rectal cancer cell lines, although the mechanism is poorly understood. We hypothesized that ST6Gal-1 increases following chemoradiation in primary rectal cancer organoids similar to what we have found in cell lines and that the mechanism of resistance is via decreased apoptosis.

Methods: We assessed ST6Gal-1 in treated rectal cancer organoids made from patient tumor biopsies using fluorescent staining and PCR. SW620 cells were transduced with lentivirus containing shST6Gal-1 to knockdown the mRNA (KD) or an shRNA control vector (CV). Flow cytometry was used to assess Annexin V as a measure of apoptosis. SNA is a lectin which recognizes sialic acid placed on proteins by ST6Gal-1. An SNA Lectin pulldown was performed, and western blotting was used to assess for TNFR1 antibody.

Results: We treated primary rectal cancer with chemoradiation and assessed for ST6GaL-1. We found that ST6Gal-1 protein increased via immunofluorescent staining and PCR revealed a 3-fold increase in ST6Gal-1 mRNA on day 5 (N=3, p=0.007). To further explore mechanism, flow cytometry was performed in SW 620 rectal cancer cells following chemoradiation and identified 33.3% apoptotic cells in the KD group compared to 18.66% in CV cells containing ST6Gal-1. TNFR1, which has previously been shown to be sialylated, was also explored as a potential target. Cells underwent SNA lectin pulldown to collect sialylated proteins and found sialylated TNFR1 increased 2.91-fold in treated CV samples compared to untreated but this difference was not seen in KD samples.

We have shown that ST6Gal-1 is increased in cell lines and rectal cancer organoids after chemoradiation, and that apoptosis is likely one mechanism mediating this resistance via TNFR1 sialylation.