Mechanistic Insights of GCNT3 in PC Pathogenesis

Background: In one of my earlier projects, I conducted top-down transcriptomic analysis and identified from 200 enzymes, around 7 key glycogenes important in Pancreatic Cancer (PC) progression and metastasis. From this panel, GCNT3 stood out as one member important due to its function of adding Core-2 glycan structure onto the O-linked glycosylation. I have performed a preliminary study on this glycogene in the context of PC and thus think that it will be wise to explore its function in greater depth. Another article by Rao et al on GCNT3 exhibits differential expression in normal versus PC conditions but lacks mechanistic insights that may be involved in cellular transmigration onto endothelium. Thus, the role of GCNT3 with an emphasis on the β-catenin/MUC4 axis may be explored. To study this, the following aims may suffice:

Aim 1: Insilico analysis to illustrate correlation between GCNT3, β-catenin and mucins

For this aim, employ various online cancer databases such as cbioportal, R2, mipanda etc. and develop correlation between GCNT3, β-catenin and mucinous proteins. Also perform Gene Set Enrichment Analysis (GSEA) on normal and PC dataset from GEO portal and identify if GCNT3 is differentially expressed in PC patients along with other genes such as β-catenin and mucins. On establishing this, conduct immunohistochemistry onto different stages of PC towards the identification of GCNT3, β-catenin and mucins expressional variation.

Aim 2: Functional characterization of GCNT3 knockout (KO) and overexpression in PC cells

Here, use CRISPR/Cas9 to KO GCNT3 in CD18 and Capan-1 PC cell lines. Use CRISPR/Cas9 edited cells for quantitative estimation of cell cycle, EMT, stem cells, wnt/β-catenin and mucin pathways. Also conduct wound healing, colony formation, and transwell migration assays to look for the in vitro effect of GCNT3 in PC progression and metastasis. Further, overexpress GCNT3 in CD18 and Capan-1 PC cells to identify if the reversal of the above genetic and phenotypic effects occurs.

Aim 3: Development of metastatic CD18 and Capan-1 cell lines and illustration of GCNT3 mechanism in PC

Here, culture CD18 and Capan-1 cell lines into transwell migration chamber and carefully isolate cells that have migrated from boyden chamber. Sub-culture these cells to generate metastatic cell lines. Once the metastatic cell line is obtained, expression of glyco- and non-glyco genes can be evaluated using immune-, lectin- and mucin-blots. Use these cell lines and inject into immunocompromised mice to generate xenograft model. Pharmacological GCNT3 inhibitor, along with the first-line therapy for PC, may be injected into mice to study in vivo tumor progression and metastatic process. Moreover, develop 3D organoids using Capan-1 and CD18 isolated cancer stem cells to evaluate the efficacy of GCNT3 inhibitor along with the first-line therapy for PC.