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

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.

C1GALT1 induces the carcinogenesis of thyroid cancer through regulation by miR-141-3p and GLUT1

“…Lin explained that the loss of C1GALT1 inhibited tumor growth in the mouse models of head and neck cancer [ 23 ]. Other studies also demonstrated that C1GALT1 was used to as a biomarker to identify different cancers [ [21] , [24] , [25] ]. C1GALT1 levels were consistent with FGFR3 expression levels, a specific bladder protein, in bladder cancer [ 21 ].…”

“…C1GALT1 levels were consistent with FGFR3 expression levels, a specific bladder protein, in bladder cancer [ 21 ]. Depletion of C1GALT1 also induced the expression of invasive proteins in endometrial cancer, increasing the invasion ability of cancer cells [ 25 ]. To date, this is the first study to declare the positive regulatory relationship between C1GALT1 and GLUT1.…”

Unraveling the role of C1GALT1 in abnormal glycosylation and colorectal cancer progression

“…A recent study on endometrial cancer showed that low expression of C1GALT1 induced overexpression of ANXA1 in ECC-1 cells, which were characterized by higher proliferation, invasion, migration, colony formation and angiogenesis ( 79 ). Besides, C1GALT1 is able to modify O-linked glycosylation on integrin α5, thereby modulating activation of the PI3K/AKT pathway in gastric cancer cells ( 80 ). Also in pancreatic cancer, C1GALT1 knockdown significantly inhibited cell adhesion to the extracellular matrix (ECM), which was associated with a decrease in FAK phosphorylation at Y397/Y925 as well as changes in O-glycans on integrins (including β1, αv and α5 subunits) ( 81 ).…”

Functional Proteomics Characterization of the Role of SPRYD7 in Colorectal Cancer Progression and Metastasis

“…SPRYD7 and control siRNAs were purchased from Sigma (EHU133421 and SIC001, respectively). For siRNA transfections, 2.5 × 10 5 cells were seeded in culture plates and maintained in DMEM with 10% fetal bovine serum at 37 • C in 5% CO 2 for 24 h, according to established protocols [18,21]. Then, cells were transfected with 22 pmol siRNA using 2 µL JetPrime Transfection reagent in 100 µL of JetPrime buffer.…”

“…Gain-of-function cell-based assays were performed with CRC cells stably overexpressing SPRYD7, with mock-stably transfected cells as control, as previously described [14,18,21]. Alternatively, loss-of-function cell-based assays were performed 24 h after transient transfection of CRC cells with SPRYD7 or control siRNAs [22].…”