ST6GAL1 sialyltransferase promotes acinar to ductal metaplasia and pancreatic cancer progression
Nikita Bhalerao, Asmi Chakraborty, Michael P. Marciel, Jihye Hwang, Colleen M. Britain, Austin D. Silva, Isam E. Eltoum, Robert B. Jones, Katie L. Alexander, Lesley E. Smythies, Phillip D. Smith, David K. Crossman, Michael R. Crowley, Boyoung Shin, Laurie E. Harrington, Zhaoqi Yan, Maigen M. Bethea, Chad S. Hunter, Christopher A. Klug, Donald J. Buchsbaum, Susan L. Bellis
Nikita Bhalerao, Asmi Chakraborty, Michael P. Marciel, Jihye Hwang, Colleen M. Britain, Austin D. Silva, Isam E. Eltoum, Robert B. Jones, Katie L. Alexander, Lesley E. Smythies, Phillip D. Smith, David K. Crossman, Michael R. Crowley, Boyoung Shin, Laurie E. Harrington, Zhaoqi Yan, Maigen M. Bethea, Chad S. Hunter, Christopher A. Klug, Donald J. Buchsbaum, Susan L. Bellis
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Research Article Oncology

ST6GAL1 sialyltransferase promotes acinar to ductal metaplasia and pancreatic cancer progression

Abstract

The role of aberrant glycosylation in pancreatic ductal adenocarcinoma (PDAC) remains an under-investigated area of research. In this study, we determined that ST6 β-galactoside α2,6 sialyltransferase 1 (ST6GAL1), which adds α2,6-linked sialic acids to N-glycosylated proteins, was upregulated in patients with early-stage PDAC and was further increased in advanced disease. A tumor-promoting function for ST6GAL1 was elucidated using tumor xenograft experiments with human PDAC cells. Additionally, we developed a genetically engineered mouse (GEM) model with transgenic expression of ST6GAL1 in the pancreas and found that mice with dual expression of ST6GAL1 and oncogenic KRASG12D had greatly accelerated PDAC progression compared with mice expressing KRASG12D alone. As ST6GAL1 imparts progenitor-like characteristics, we interrogated ST6GAL1’s role in acinar to ductal metaplasia (ADM), a process that fosters neoplasia by reprogramming acinar cells into ductal, progenitor-like cells. We verified ST6GAL1 promotes ADM using multiple models including the 266-6 cell line, GEM-derived organoids and tissues, and an in vivo model of inflammation-induced ADM. EGFR is a key driver of ADM and is known to be activated by ST6GAL1-mediated sialylation. Importantly, EGFR activation was dramatically increased in acinar cells and organoids from mice with transgenic ST6GAL1 expression. These collective results highlight a glycosylation-dependent mechanism involved in early stages of pancreatic neoplasia.

Authors

Nikita Bhalerao, Asmi Chakraborty, Michael P. Marciel, Jihye Hwang, Colleen M. Britain, Austin D. Silva, Isam E. Eltoum, Robert B. Jones, Katie L. Alexander, Lesley E. Smythies, Phillip D. Smith, David K. Crossman, Michael R. Crowley, Boyoung Shin, Laurie E. Harrington, Zhaoqi Yan, Maigen M. Bethea, Chad S. Hunter, Christopher A. Klug, Donald J. Buchsbaum, Susan L. Bellis

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Figure 5

ST6GAL1 activity enhances the initiation and growth of GEM-derived organoids and promotes changes in gene expression consistent with a more progenitor-like state.

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ST6GAL1 activity enhances the initiation and growth of GEM-derived organ...
(A) Cells dissociated from WT organoids were seeded into either organoid (ORG) culture, which maintains stemness properties, or monolayer (ML) culture in media with reduced stem cell factors, which induces cell differentiation. qRT-PCR analyses revealed that, similar to the stemness genes Axin2 and Lgr5, endogenous St6gal1 mRNA expression is downregulated in ML cultures (n = 3). Data analyzed using a Student’s t test. *P < 0.05. qRT-PCR, quantitative reverse transcriptase PCR; RQ, relative quantification. (B) Cells were dissociated from organoid lines and 2,000 cells seeded into fresh organoid culture. The number of organoids formed at day 3 was enumerated (n = 3 independent experiments, with 3 wells per genotype counted). Data analyzed by 1-way ANOVA, *P < 0.05. (C) Cells were dissociated from organoids, then seeded into fresh organoid culture, and organoid growth was monitored over time. Scale bar: 100 μm. (D) At days 3, 6, and 9 following the seeding of 2,000 organoid-derived cells into fresh organoid culture, organoids were dissociated, and the total number of cells was quantified (n = 4). Data analyzed using a Student’s t test. *P < 0.05. (E) Sox9, Hes1, and Ptf1a mRNA was quantified by qRT-PCR (n = 3). Data analyzed by 1-way ANOVA. *P < 0.05. (F) Organoids were lysed and immunoblotted for SOX9 and ST6GAL1. Densitometric analyses were conducted on blots from 4 independent organoid lysates. Data analyzed by 1-way ANOVA. *P < 0.05. Relative D.U., densitometric units normalized to β-tubulin. (G) RNA-Seq was conducted on organoid lines generated from 3 distinct mice per genotype. The heatmap depicts canonical acinar and ductal genes. Color key indicates z score.