ST6GalNAc-I regulates tumor cell sialylation via NECTIN2/MUC5AC-mediated immunosuppression and angiogenesis in non–small cell lung cancer
Muthamil Iniyan Appadurai, Sanjib Chaudhary, Ashu Shah, Gopalakrishnan Natarajan, Zahraa W. Alsafwani, Parvez Khan, Dhananjay D. Shinde, Subodh M. Lele, Lynette M. Smith, Mohd Wasim Nasser, Surinder Kumar Batra, Apar Kishor Ganti, Imayavaramban Lakshmanan
Muthamil Iniyan Appadurai, Sanjib Chaudhary, Ashu Shah, Gopalakrishnan Natarajan, Zahraa W. Alsafwani, Parvez Khan, Dhananjay D. Shinde, Subodh M. Lele, Lynette M. Smith, Mohd Wasim Nasser, Surinder Kumar Batra, Apar Kishor Ganti, Imayavaramban Lakshmanan
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Research Article Cell biology Immunology Oncology

ST6GalNAc-I regulates tumor cell sialylation via NECTIN2/MUC5AC-mediated immunosuppression and angiogenesis in non–small cell lung cancer

Abstract

Glycosylation controls immune evasion, tumor progression, and metastasis. However, how tumor cell sialylation regulates immune evasion remains poorly characterized. ST6GalNAc-I, a sialyltransferase that conjugates sialic acid to the glycans in glycoproteins, was overexpressed in an aggressive-type KPA (KrasG12D/+ Trp53R172H/+ Ad-Cre) lung adenocarcinoma (LUAD) model and patient samples. Proteomic and biochemical analysis indicated that ST6GalNAc-I mediated NECTIN2 sialylation in LUAD cells. ST6GalNAc-I–deficient tumor cells cocultured with T cells were more susceptible to T cell–mediated tumor cell killing, indicating a key role for NECTIN2 in T cell dysfunction. Mice injected with St6galnac-I–knockdown syngeneic cells showed reduced lung tumor incidence and Nectin2/Tigit-associated immunosuppression. ST6GalNAc-I–deficient cells exhibited reduced P-DMEA metabolite levels, while administration of P-DMEA promoted LUAD cell proliferation via MUC5AC. MUC5AC interacted and colocalized with PRRC1 in the Golgi, suggesting a potential role for PRRC1 in MUC5AC glycosylation. Mice injected with ST6GalNAc-I/MUC5AC-deficient cells (human LUAD) exhibited reduced lung tumor incidence, angiogenesis, and liver metastases. Mechanistically, ST6GalNAc-I/MUC5AC regulates VCAN-V1, a key factor in tumor matrix remodeling during angiogenesis and metastasis. These findings demonstrate that ST6GalNAc-I–mediated sialylation of NECTIN2/MUC5AC is critical for immune evasion and tumor angiogenesis. Targeting this pathway may prevent LUAD development and/or metastasis.

Authors

Muthamil Iniyan Appadurai, Sanjib Chaudhary, Ashu Shah, Gopalakrishnan Natarajan, Zahraa W. Alsafwani, Parvez Khan, Dhananjay D. Shinde, Subodh M. Lele, Lynette M. Smith, Mohd Wasim Nasser, Surinder Kumar Batra, Apar Kishor Ganti, Imayavaramban Lakshmanan

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Figure 6

ST6GalNAc-I and MUC5AC promote proliferation and endothelial cell migration.

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ST6GalNAc-I and MUC5AC promote proliferation and endothelial cell migrat...
(A) Schematic showing coculture for the angiogenesis experiments. (B) Western blot analysis indicated expression of MUC5AC in various LUAD cells but not in endothelial cells. (C and D) IncuCyte-based proliferation and tube formation ability of HULEC-5a cells (labeled with CellTracker Orange CMTMR Dye) cocultured with A549 ST6GalNAc-I–KO or A549 MUC5AC-KD cells in Matrigel-coated plates. ST6GalNAc-I–KO and A549 MUC5AC-KD cells showed poor tube formation ability and proliferation. Significance was determined by 2-tailed t test (n = 3). Scale bars: 400 μm. (E) Schematic showing experimental design for migration assay. (F) Transwell migration assay of HULEC-5a cells with A549 ST6GalNAc-I–KO and MUC5AC-KD cell–derived conditioned medium and respective control cells. Data were analyzed using 2-tailed t test. Scale bars: 400 μm. (G) MUC5AC sandwich ELISA in secretome (conditioned media) of A549 ST6GalNAc-I–KO and A549 MUC5AC-KD cells. Significance was determined by 2-tailed t test (n = 3). (H and I) GSEA was done in pre-ranked differentially expressed genes of MUC5AC-KD versus scramble. MUC5AC-KD genes were significantly associated with endothelial cell proliferation and migration. (J) Metabolomic analysis was performed using A549 ST6GalNAc-I–KO and control cells. (K) Heatmap represents differential expression of metabolites between A549 ST6GalNAc-I–KO and control cells using MetaboAnalyst 6.0. (L) P-DMEA–treated (100 μM) ST6GalNAc-I–KO (endogenously low P-DMEA) cells showed increased proliferation properties, suggesting that P-DMEA may be required for LUAD cell growth. Statistical significance was determined by linear mixed model (LMM) (n = 3) at 44 hours (Padj = 0.04). (M) P-DMEA–treated shMUC5AC cells showed significantly increased proliferation after 40–64 hours (Padj < 0.01), suggesting that P-DMEA may regulate LUAD cell growth through MUC5AC. Significance was determined by LMM (n = 8). Padj, P value adjusted with the Westfall stepdown method.