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  3. In-depth quantitative proteomics analysis revealed C1GALT1 depletion in ECC-1 cells mimics an aggressive endometrial cancer phenotype observed in cancer patients with low C1GALT1 expression

In-depth quantitative proteomics analysis revealed C1GALT1 depletion in ECC-1 cells mimics an aggressive endometrial cancer phenotype observed in cancer patients with low C1GALT1 expression

  • Cell Oncol (Dordr). 2023 Feb 6. doi: 10.1007/s13402-023-00778-w.
Ana Montero-Calle 1,  Álvaro López-Janeiro 2,  Marta L Mendes 3,  Daniel Perez-Hernandez 3,  Irene Echevarría 1 4,  Ignacio Ruz-Caracuel 2,  Victoria Heredia-Soto 5 6,  Marta Mendiola 6 7,  David Hardisson 2 6 7 8,  Pablo Argüeso 9,  Alberto Peláez-García 10,  Ana Guzman-Aranguez 11,  Rodrigo Barderas 12 13
Affiliations

Affiliations

  • 1 Chronic Disease Programme, UFIEC, Instituto de Salud Carlos III, 28220, Majadahonda, Madrid, Spain.
  • 2 Department of Pathology, Hospital Universitario La Paz, 28046, Madrid, Spain.
  • 3 Department of Infection and Immunity, Luxembourg Institute of Health, 1445, Strassen, Luxembourg.
  • 4 Biochemistry and Molecular Biology Department, Facultad de Óptica y Optometría, Universidad Complutense de Madrid, 28037, Madrid, Spain.
  • 5 Translational Oncology, La Paz University Hospital (IdiPAZ), 28046, Madrid, Spain.
  • 6 Center for Biomedical Research in the Cancer Network (Centro de Investigación Biomédica en Red de Cáncer, CIBERONC), Instituto de Salud Carlos III, 28046, Madrid, Spain.
  • 7 Molecular Pathology and Therapeutic Targets Group, La Paz University Hospital (IdiPAZ), 28046, Madrid, Spain.
  • 8 Faculty of Medicine, Universidad Autónoma de Madrid, 28029, Madrid, Spain.
  • 9 Tufts Medical Center, Tufts University School of Medicine, Boston, Massachusetts, USA.
  • 10 Molecular Pathology and Therapeutic Targets Group, La Paz University Hospital (IdiPAZ), 28046, Madrid, Spain. alberto.pelaez@idipaz.es.
  • 11 Biochemistry and Molecular Biology Department, Facultad de Óptica y Optometría, Universidad Complutense de Madrid, 28037, Madrid, Spain. aguzman@opt.ucm.es.
  • 12 Chronic Disease Programme, UFIEC, Instituto de Salud Carlos III, 28220, Majadahonda, Madrid, Spain. r.barderasm@isciii.es.
  • 13 Functional Proteomics Unit, UFIEC, Instituto de Salud Carlos III, 28220, Majadahonda, Madrid, Spain. r.barderasm@isciii.es.
PMID: 36745330 DOI: 10.1007/s13402-023-00778-w
Abstract

Background: Endometrial Cancer (EC) is the most common Cancer of the female reproductive organs. Despite the good overall prognosis of most low-grade ECs, FIGO I and FIGO II patients might experience tumor recurrence and worse prognosis. The study of alterations related to EC pathogenesis might help to get insights into underlying mechanisms involved in EC development and progression.

Methods: Core tumoral samples were used to investigate the role of C1GALT1 in EC by immunohistochemistry (IHC). ECC-1 cells were used as endometrioid EC model to investigate the effect of C1GALT1 depletion using C1GALT1 specific shRNAs. SILAC quantitative proteomics analyses and cell-based assays, PCR, qPCR, WB, dot-blot and IHC analyses were used to identify, quantify and validate dysregulation of proteins.

Results: Low C1GALT1 protein expression levels associate to a more aggressive phenotype of EC. Out of 5208 proteins identified and quantified by LC-MS/MS, 100 proteins showed dysregulation (log2fold-change ≥ 0.58 or ≤-0.58) in the cell protein extracts and 144 in the secretome of C1GALT1 depleted ECC-1 cells. Nine dysregulated proteins were validated. Bioinformatics analyses pointed out to an increase in pathways associated with an aggressive phenotype. This finding was corroborated by loss-of-function cell-based assays demonstrating higher proliferation, invasion, migration, colony formation and angiogenesis capacity in C1GALT1 depleted cells. These effects were associated to the overexpression of ANXA1, as demonstrated by ANXA1 transient silencing cell-based assays, and thus, correlating C1GALT and ANXA1 protein expression and biological effects. Finally, the negative protein expression correlation found by proteomics between C1GALT1 and LGALS3 was confirmed by IHC.

Conclusion: C1GALT1 stably depleted ECC-1 cells mimic an EC aggressive phenotype observed in patients and might be useful for the identification and validation of EC markers of progression.

Keywords

C1GALT1; Endometrial cancer; O-glycosylation; Quantitative proteomics; SILAC.

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