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Terminal α2,6-sialylation of epidermal growth factor receptor modulates antibody therapy response of colorectal cancer cells

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Abstract

Background

The epidermal growth factor receptor (EGFR) is a key protein involved in cancer development. Monoclonal antibodies targeting EGFR are approved for the treatment of metastatic colorectal cancer (CRC). Despite the beneficial clinical effects observed in subgroups of patients, the acquisition of resistance to treatment remains a major concern. Protein N-glycosylation of cellular receptors is known to regulate physiological processes leading to activation of downstream signaling pathways. In the present study, the role of EGFR-specific terminal ⍺2,6-sialylation was analyzed in modulation of the malignant phenotype of CRC cells and their resistance to monoclonal antibody Cetuximab-based therapy.

Methods

Glycoengineered CRC cell models with specific sialyltransferase ST6GAL1 expression levels were applied to evaluate EGFR activation, cell surface glycosylation and therapeutic response to Cetuximab.

Results

Glycoproteomic analysis revealed EGFR as a major target of ST6Gal1-mediated ⍺2,6-sialylation in a glycosite-specific manner. Mechanistically, CRC cells with increased ST6Gal1 expression and displaying terminal ⍺2,6-sialylation showed a marked resistance to Cetuximab-induced cytotoxicity. Moreover, we found that this resistance was accompanied by downregulation of EGFR expression and its activation.

Conclusions

Our data indicate that EGFR ⍺2,6-sialylation is a key factor in modulating the susceptibility of CRC cells to antibody targeted therapy, thereby disclosing a potential novel biomarker and providing key molecular information for tailor made anti-cancer strategies.

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Data availability

The mass spectrometry proteomic data supporting the conclusions of this article are available in the ProteomeXchange Consortium via the PRIDE partner repository, under the project name “EGFR N-Glycosylation Profile from Colorectal Cancer Cells”, with the dataset identifier PXD017914.

Code availability

Not applicable.

Abbreviations

⍺2,3NeuAc:

⍺2,3-linked sialic acid

⍺2,6NeuAc:

⍺2,6-linked sialic acid

AAL:

Aleuria aurantia lectin

BrdU:

bromodeoxyuridine

CRC:

colorectal cancer

CRISPR/Cas9:

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9

ECL:

enhanced chemiluminescence

EGFR:

epidermal growth factor receptor

FBS:

fetal bovine serum

gRNA:

guide RNA

IDAA:

Indel Detection by Amplicon Analysis

Indel:

insertions and deletions

KO:

Knock-Out

LTL:

Lotus tetragonolobus lectin

mAb:

monoclonal antibody

MAL-I:

Maackia amurensis lectin I

MFI:

median fluorescence intensity

OE:

Overexpression

ON:

overnight

RT:

room temperature

RTK:

receptor tyrosine kinase

SNA:

Sambucus nigra agglutinin

ST6Gal1:

β-Galactoside ⍺2,6-sialyltransferase 1

TIDE:

Tracking of Indels by DEcomposition

TNFR1:

tumor necrosis receptor 1

UEA-I:

Ulex europaeus agglutinin I

WT:

wild-type

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Acknowledgements

The authors acknowledge Merck KGaA, Darmstadt, Germany for providing the anti-Cetuximab and anti-Matuzumab antibodies. The authors acknowledge Dr. Anne Harduin-Lepers and Dr. Virginie Cogez for their help with the preparation of the pcDNA3.1/Hygro(+)ST6Gal1 plasmid. The authors acknowledge Dr. Luis Cirnes from Ipatimup Diagnostics for the support on the analysis of cell line genetic statuses. The authors acknowledge the support of the i3S Advanced Light Microscopy facility, member of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122) and the i3S Proteomics Scientific Platform, member of the Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance (ROTEIRO/0028/2013; LISBOA-01-0145-FEDER-0221 25). The authors acknowledge the i3S Translational Research and Industry Partnership Office for all the support given for the scientific design and for the continuous monitoring of the project.

Funding

This research was funded by Merck KGaA, Darmstadt, Germany, and by FEDER funds through the Operational Programme for Competitiveness Factors COMPETE 2020 (POCI-01-0145-FEDER-016585; POCI-01-0145-FEDER-007274) and national funds through the Foundation for Science and Technology (FCT), under the projects: PTDC/BBB-EBI/0567/2014 to C.A.R and UID/BIM/04293/2013; PTDC/MED-QUI/29,780/2017 to C.G., and the project NORTE-01-0145-FEDER-000029, supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). J.G.R. was supported by a FCT PhD grant (SFRH/BD/136,736/2018); H.O.D. was supported by a FCT PhD grant (PD/BD/128,407/2017) through the FCT PhD Programmes and by Programa Operacional Potencial Humano (POPH), specifically by the BiotechHealth Programme (Doctoral Programme on Cellular and Molecular Biotechnology Applied to Health Sciences), with the reference PD/0016/2012 funded by FCT. M.B. was supported by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement n.º 748,880.

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Author notes

  1. Meritxell Balmaña

    Present address: Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), 1030, Vienna, Austria

  2. Joana Gomes and Celso A. Reis contributed equally to this work.

Authors and Affiliations

  1. i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135, Porto, Portugal

    Joana G. Rodrigues, Henrique O. Duarte, Catarina Gomes, Meritxell Balmaña, Álvaro M. Martins, Jorge Lima, André Albergaria, Joana Gomes & Celso A. Reis

  2. IPATIMUP – Institute of Molecular Pathology and Immunology of the University of Porto, 4200-135, Porto, Portugal

    Joana G. Rodrigues, Henrique O. Duarte, Catarina Gomes, Meritxell Balmaña, Álvaro M. Martins, Jorge Lima, André Albergaria, Joana Gomes & Celso A. Reis

  3. Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050-313, Porto, Portugal

    Joana G. Rodrigues, Henrique O. Duarte & Celso A. Reis

  4. Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands

    Paul J. Hensbergen, Arnoud H. de Ru, Peter A. van Veelen & Manfred Wuhrer

  5. Faculty of Medicine, University of Porto, 4200-319, Porto, Portugal

    Jorge Lima, André Albergaria & Celso A. Reis

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  1. Joana G. Rodrigues
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J.G. and C.A.R. designed and supervised the study. J.G.R., H.O.D., C.G., M.B., A.M., A.H.d.R. and J.G performed experiments; J.G.R., H.O.D., C.G., P.J.H., J.L., A.A., P.A.V., M.W., J.G. and C.A.R performed the formal analyses; J.G.R., H.O.D., J.G. and C.A.R wrote the original manuscript. All authors reviewed and edited the manuscript. All authors have read and agreed to the final version of the manuscript.

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Correspondence to Joana Gomes or Celso A. Reis.

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Supplementary Information

ESM 1

Fig. S1 Genomic validation of CRISPR/Cas9 ST6GAL1 KO in Caco-2 cell line. a Validation of nucleotide insertions and deletions (indels) at the ST6GAL1 locus following genome editing by CRISPR/Cas9, in three isogenic Caco-2 ST6Gal1 KO cell clones (KO 17, KO 33 and KO 42) by Indel Detection by Amplicon Analysis (IDAA) PCR. b Validation of Caco-2 ST6Gal1 KO cell clones indels using the Tracking of Indels by DEcomposition (TIDE) bioinformatic tool. Fig. S2 ST6Gal1 overexpression induces the enrichment of EGFR N444 and N530 glycosites in terminally sialylated species. a Upper panel - higher energy collision dissociation (HCD) MS/MS spectra of the N444 glycopeptide modified with a mono-sialylated biantennary N-glycan in SW48 ST6Gal1 OE 1 EGFR; Middle panel – collision-induced dissociation (CID) MS/MS spectrum of the same glycopeptide; Bottom panel – HCD MS/MS spectra of the same glycopeptide depicting identified b and y ions for peptide identification. b Upper panel - HCD MS/MS spectra of the N528 glycopeptide modified with a di-sialylated biantennary N-glycan in SW48 ST6Gal1 OE 1 EGFR; Middle panel – CID MS/MS spectrum of the same glycopeptide; Bottom panel – HCD MS/MS spectra of the same glycopeptide depicting identified b and y ions for peptide identification. Fig. S3 EGFR site-specific glycan composition in Caco-2 cells. a Detection of ⍺2,6-linked sialic acid (⍺2,6NeuAc) structures in immunoprecipitated EGFR from Caco-2 WT and ST6Gal1 KO (KO 17, KO 33 and KO 42) cell clones by reactivity with SNA. b Colloidal Blue gel staining of immunoprecipitated EGFR from Caco-2 WT and ST6Gal1 KO cell clones. c Schematic representation of EGFR glycosylation following site-specific assignment and structural glycan characterization in Caco-2 WT and ST6Gal1 KO cell clones. Fig. S4. Impact of ST6Gal1 overexpression in SW48 cells proliferation, cell death and binding to cetuximab in the absence of treatment. a Proliferation quantification in untreated SW48 WT, Mock and ST6Gal1 OE clones (OE 1 and OE 3) by flow cytometry analysis of bromodeoxyuridine (BrdU) staining. b Quantification of cell death by Annexin V/Propidium Iodate apoptosis assay in untreated SW48 WT, Mock and ST6Gal1 OE cell clones. c Cetuximab binding to cell surface EGFR in untreated SW48 WT, Mock and ST6Gal1 OE cell clones. Comparisons between multiple groups were made using one-way ANOVA, with Mock as control group. (PDF 2.62 MB)

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Rodrigues, J.G., Duarte, H.O., Gomes, C. et al. Terminal α2,6-sialylation of epidermal growth factor receptor modulates antibody therapy response of colorectal cancer cells. Cell Oncol. 44, 835–850 (2021). https://doi.org/10.1007/s13402-021-00606-z

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