CTNND1 is involved in germline predisposition to early-onset gastric cancer by affecting cell-to-cell interactions

Abstract

Background: CDH1 and CTNNA1 remain as the main genes for hereditary gastric cancer. However, they only explain a small fraction of gastric cancer cases with suspected inherited basis. In this study, we aimed to identify new hereditary genes for early-onset gastric cancer patients (EOGC; < 50 years old).

Methods: After germline exome sequencing in 20 EOGC patients and replication of relevant findings by gene-panel sequencing in an independent cohort of 152 patients, CTNND1 stood out as an interesting candidate gene, since its protein product (p120ctn) directly interacts with E-cadherin. We proceeded with functional characterization by generating two knockout CTNND1 cellular models by gene editing and introducing the detected genetic variants using a lentiviral delivery system. We assessed β-catenin and E-cadherin levels, cell detachment, as well as E-cadherin localization and cell-to-cell interaction by spheroid modeling.

Results: Three CTNND1 germline variants [c.28_29delinsCT, p.(Ala10Leu); c.1105C > T, p.(Pro369Ser); c.1537A > G, p.(Asn513Asp)] were identified in our EOGC cohorts. Cells encoding CTNND1 variants displayed altered E-cadherin levels and intercellular interactions. In addition, the p.(Pro369Ser) variant, located in a key region in the E-cadherin/p120ctn binding domain, showed E-cadherin mislocalization.

Conclusions: Defects in CTNND1 could be involved in germline predisposition to gastric cancer by altering E-cadherin and, consequently, cell-to-cell interactions. In the present study, CTNND1 germline variants explained 2% (3/172) of the cases, although further studies in larger external cohorts are needed.

Keywords: Cell adhesion; Delta catenin; Early-onset disease; Genetic predisposition to disease; Stomach neoplasms.

Fig. 1

Functional characterization of the CTNND1-depleted NCI-N87 cell model by CRISPR/Cas9. a Quantification of relative protein levels of E-cadherin and β-catenin by Western blot. GAPDH was used as internal control. Representative blot of n = 4 (mean ± SD; Welch’s t-test, ^∗∗∗P < 0.001). b Immunofluorescent staining of spheroids (NCI-N87 and NCI KO10) using p120ctn and E-cadherin antibodies. CRISPR-mediated CTNND1 editing resulted in p120ctn complete depletion and reduced E-cadherin expression. Wide-field microscopy images (left panel) were captured using a 10 × objective (Scale bar = 100 µm). A small area is shown at higher magnification (right panel, 63 × oil objective, scale bar = 20 µm). c Representative pictures of NCI-N87 and NCI KO10 spheroids. Scale bar = 200 µm. Circularity, compactness and roundness index decreased after p120ctn deletion. Spheroids were generated at least in quintuplicate and the experiment was repeated 3 times (n = 3; mean ± SD; Welch’s t-test, ^∗∗∗P < 0.001). d Detachment time-course assay. Data represent the percentage of remaining attached cells after being treated with trypsin at different timepoints (n = 9, mean ± SD; Welch’s t-test, ^∗P < 0.05)

Fig. 2

CTNND1 variants compromise E-cadherin levels and its membrane localization. a Quantification of relative protein levels of E-cadherin and β-catenin by Western blot. GAPDH was used as internal control. Representative blot of n = 4 (mean ± SD; analysis of variance with LSD post hoc test, ^*P < 0.05). b Immunofluorescent staining of spheroids expressing p120ctn variants. The vesicular internalization pattern of E-cadherin can be detected in cells expressing CTNND1 p.(Pro369Ser). Wide-field microscopy images (left panel) were captured using a 10 × objective (Scale bar = 100 µm). A small area is shown at higher magnification (right panel, 63 × oil objective, scale bar = 20 µm). Protein annotation is in its short form

Fig. 3

CTNND1 variants affect cell-to-cell interactions. a Representative pictures of spheroids expressing each CTNND1 variant or its wild-type counterpart. Circularity, compactness and roundness index are represented. Spheroids were generated at least in quintuplicate and the experiment was repeated 3 times (n = 3; mean ± SD; analysis of variance with LSD post hoc test, ^∗P < 0.05). b Detachment assay. Data represent the percentage of remaining attached cells after being treated with trypsin for 8 min (n = 9, mean ± SD; analysis of variance with LSD post hoc test, ^∗P < 0.05). Protein annotation is in its short form

Fig. 4

Schematic representation of p120ctn variants within the functional domains of the protein. ARM Armadillo domain

Fig. 5

Schematic representation of the proposed molecular mechanism of pathogenicity for CTNND1 germline variants. CTNND1 germline variants affect p120ctn-E-cadherin binding and compromise cell-to-cell interactions, which could lead to GC predisposition