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Reduction in O-glycome induces differentially glycosylated CD44 to promote stemness and metastasis in pancreatic cancer

Oncogene volume 41, pages 57–71 (2022)Cite this article

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Abstract

Aberrant protein glycosylation has been shown to have a significant contribution in aggressive cancer, including pancreatic cancer (PC). Emerging evidence has implicated the involvement of cancer stem cells (CSCs) in PC aggressiveness; however, the contribution of glycosylation on self-renewal properties and maintenance of CSC is understudied. Here, using several in vitro and in vivo models lacking C1GALT1 expression, we identified the role of aberrant O-glycosylation in stemness properties and aggressive PC metastasis. A loss in C1GALT1 was found to result in the truncation of O-glycosylation on several glycoproteins with an enrichment of Tn carbohydrate antigen. Mapping of Tn-bearing glycoproteins in C1GALT1 KO cells identified significant Tn enrichment on CSC glycoprotein CD44. Notably, a loss of C1GALT1 in PC cells was found to enhance CSC features (side population-SP, ALDH1+, and tumorspheres) and self-renewal markers NANOG, SOX9, and KLF4. Furthermore, a loss of CD44 in existing C1GALT1 KO cells decreased NANOG expression and CSC features. We determined that O-glycosylation of CD44 activates ERK/NF-kB signaling, which results in increased NANOG expression in PC cells that facilitated the alteration of CSC features, suggesting that NANOG is essential for PC stemness. Finally, we identified that loss of C1GALT1 expression was found to augment tumorigenic and metastatic potential, while an additional loss of CD44 in these cells reversed the effects. Overall, our results identified that truncation of O-glycans on CD44 increases NANOG activation that mediates increased CSC activation.

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Fig. 1: Mass spectrometry results identify CD44 as a marker of aberrant glycosylation in PC.
Fig. 2: The loss of C1GALT1 enhances self-renewal markers and features of CSCs.
Fig. 3: CD44 is heavily o-linked with Tn-antigens in aberrant glycosylation of PC.
Fig. 4: Truncation of Tn-antigen o-linked CD44 increases CSC features via NF-κB signaling.
Fig. 5: Tn-antigen truncation of CD44 elevates CSC features and NANOG expression via NF-kB signaling in PC cells.
Fig. 6: O-linked truncation of CD44 enhances in vitro and in vivo tumorigenesis.
Fig. 7: O-glycan truncation of CD44 promotes metastasis of PC.

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Acknowledgements

We thank Craig Semerad, Victoria B. Smith, and Samantha Wall of the Flow Cytometry Research Facility, University of Nebraska Medical Center, for assisting with flow cytometry. We thank Janice A. Taylor and James R. Talaska of the Advanced Microscopy Core Facility at the University of Nebraska Medical Center for assisting with confocal microscopy. Further, we thank Dr. Vikas Kumar and Dr. Dragana Lagundžin, of the Mass Spectrometry and Proteomics Core Facility at the University of Nebraska Medical Center, for assisting with proteomics. Lastly, we thank Corinn E. Grabow for assistance in technical support. We were supported primarily by the following grants from the National Institutes of Health R01 CA210637, R01 CA206444, P01 CA217798, U01 CA200466, and U01 CA210240. The authors/work in this article were supported, in parts, by the following grants from the National Institutes of Health (R01 CA210637, R01 CA206444, P01 CA217798, U01 CA200466, and U01 CA210240).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA

    Frank Leon, Parthasarathy Seshacharyulu, Rama K. Nimmakayala, Seema Chugh, Saswati Karmakar, Palanisamy Nallasamy, Raghupathy Vengoji, Satyanarayana Rachagani, Kavita Mallya, Surinder K. Batra & Moorthy P. Ponnusamy

  2. Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA

    Jesse L. Cox

  3. Eppley Institute for Research in Cancer and Allied Diseases, and Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA

    Surinder K. Batra & Moorthy P. Ponnusamy

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Contributions

MPP, SKB, and FL conceived and designed the experiments. FL performed the experiments. RKN, SC, and SK assisted with in vitro experiments. PS, PN, RV, and SR assisted with in vivo experiments. MPP, SKB, FL, and JC analyzed the data. FL and MPP wrote the manuscript.

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Correspondence to Surinder K. Batra or Moorthy P. Ponnusamy.

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SKB is one of the co-founders of Sanguine Diagnostics and Therapeutics, Inc.

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Leon, F., Seshacharyulu, P., Nimmakayala, R.K. et al. Reduction in O-glycome induces differentially glycosylated CD44 to promote stemness and metastasis in pancreatic cancer. Oncogene 41, 57–71 (2022). https://doi.org/10.1038/s41388-021-02047-2

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