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. 2022 Mar 22;14(7):1609.
doi: 10.3390/cancers14071609.

E-Cadherin-Deficient Cells Are Sensitive to the Multikinase Inhibitor Dasatinib

Nicola Bougen-Zhukov  1 , Lyvianne Decourtye-Espiard  1 , Wilson Mitchell  1 , Kieran Redpath  1 , Jacqui Perkinson  1 , Tanis Godwin  1 , Michael A Black  1 , Parry Guilford  1
Affiliations

E-Cadherin-Deficient Cells Are Sensitive to the Multikinase Inhibitor Dasatinib

Nicola Bougen-Zhukov et al. Cancers (Basel). .

Abstract

The CDH1 gene, encoding the cell adhesion protein E-cadherin, is one of the most frequently mutated genes in gastric cancer and inactivating germline CDH1 mutations are responsible for the cancer syndrome hereditary diffuse gastric cancer (HDGC). CDH1-deficient gastric cancers exhibit high AKT serine/threonine kinase 3 (AKT3) expression, but specific drugs against this AKT isoform are not available. We therefore used two publicly available datasets to identify AKT3-associated genes which could be used to indirectly target AKT3. Reactome analysis identified an enrichment of extracellular matrix remodelling genes in AKT3-high gastric cancers. Of the 51 genes that were significantly correlated with AKT3 (but not AKT1), discoidin domain receptor tyrosine kinase 2 (DDR2) showed the strongest positive association. Treatment of isogenic human cells and mouse gastric and mammary organoids with dasatinib, a small molecule inhibitor of multiple kinases including SRC, BCR-ABL and DDR2, preferentially slowed the growth and induced apoptosis of E-cadherin-deficient cells. Dasatinib treatment also preferentially slowed the growth of gastric and mammary organoids harbouring both Cdh1 and Tp53 mutations. In organoid models, dasatinib treatment was associated with decreased phosphorylation of total AKT, with a stronger effect seen in Cdh1-deficient organoids. Treatment with combinations of dasatinib and an inhibitor of AKT, MK2206, enhanced the effect of dasatinib in breast MCF10A cells. In conclusion, targeting the DDR2-SRC-AKT3 axis with dasatinib represents a promising approach for the chemoprevention and chemotherapy of gastric and breast cancers lacking E-cadherin.

Keywords: AKT serine/threonine kinase 3 AKT3; E-cadherin; HDGC; chemoprevention; dasatinib; diffuse gastric cancer; discoidin domain receptor 2 (DDR2); lobular breast cancer; synthetic lethality.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Mammary epithelial cells lacking CDH1 expression are preferentially sensitive to the cytotoxic effects of dasatinib. (A–D) Normalised MCF10a-WT and CDH1−/− cell counts 48 h after treatment with serial dilutions of dasatinib (A), imatinib mesylate (B), ponatinib (C) and nilotinib (D). Wild-type, black bars, CDH1−/− grey bars. Six fields per well at 4× magnification were captured using the Cytation 5 imager (Biotek). Nuclei were counted using Gen5 (Biotek) and normalised to the vehicle control for each cell line. (For all graphs, error bars = SEM; * p < 0.05, ** p < 0.01 and *** p <0.001; n ≥ 3 independent biological replicates; unpaired two-sided t-test).
Figure 2
Figure 2
Dasatinib induces apoptosis in mammary cells lacking E-cadherin. (A) Total apoptosis (Annexin-V-FITC and propidium iodide positive cells) (early + late apoptosis) detected by flow cytometry after 72 h drug treatment. (B) Representative histograms of MCF10A-WT and CDH1−/− cells stained with Annexin-V-FITC and propidium iodide and analysed on BD Fortessa flow cytometer. Q4; live cells, Q3; early apoptotic cells and Q2; late apoptotic cells. (For bar graph, error bars = SEM; * p < 0.05; n ≥ 3 independent biological replicates; unpaired two-sided t-test).
Figure 3
Figure 3
Characterisation of mammary organoids. (A) Endoxifen (endox) mediated knockout of E-cadherin and/or Tp53 in mammary organoids was detected utilizing western blotting. (B) Relative expression of E-cadherin and Tp53 protein in WT, Cdh1−/− and Cdh1−/−Tp53−/− mammary organoids. (C) 20× Brightfield and RFP channel images of mammary organoids induced with endoxifen.
Figure 4
Figure 4
Mouse-derived organoids containing Cdh1−/− cells are more sensitive to the growth inhibiting effects of dasatinib. Representative photos of WT and Cdh1−/− gastric (A) and mammary (B) organoids after 48 h treatment with DMSO or dasatinib. Bar graphs showing relative area of DMSO or dasatinib treated gastric (C) or mammary (D) organoids. Bar graphs showing relative area of DMSO or dasatinib treated WT or Tp53−/−Cdh1−/− gastric (E) and mammary (F) organoids. (For all graphs, error bars = SEM; * p < 0.05, ** p < 0.01 and *** p <0.001; n ≥ 3 independent biological replicates; unpaired two-sided t-test).
Figure 5
Figure 5
Dasatinib preferentially inhibits pAKT in mouse derived organoids lacking Cdh1−/−. (A,B) Western blots of pAKT-Ser473 and total AKT levels in gastric (A) and mammary (B) organoids treated with DMSO or dasatinib (0.5 µM) for 24 h. (C,D) Relative expression of pAKT gastric (C) and mammary (D) organoids. (For all graphs, error bars = SEM; * p < 0.05, ** p < 0.01; n ≥ 3 independent biological replicates; unpaired two-sided t-test).
Figure 6
Figure 6
Combining dasatinib with the allosteric AKT inhibitor MK2206 is synergistic in MCF10A cells. Normalised MCF10A-WT (A) and CDH1−/− (B) cell counts 48 h after treatment with serial dilutions of dasatinib, MK2206 or a combination of dasatinib and MK2206. (C) Combination index (CI) values for MCF10A-WT and CDH1−/− cells treated with combination of dasatinib and MK2206. Values below 0.9 indicate the drug combination is synergistic at that concentration. For all graphs, error bars = SEM; ns: p > 0.05, * p < 0.05, ** p < 0.01; n ≥ 3 independent biological replicates; unpaired two-sided t-test).
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