Targeting the Akt–EphA2 axis and cell–cell adhesion enhances anoikis sensitivity in cancer cells

Figures

• 

  Fig. 1. Tpm2.1 expression promotes anoikis in anoikis-resistant MDA-MB-231 breast cancer cells. (A) Schematic of MDA-EV and MDA-Tpm2.1 cells cultured under adherent (tissue culture plate) and suspension (low-attachment plate) conditions. (B) Representative phase-contrast images of MDA-WT, MDA-EV and MDA-Tpm2.1 cells after 8 days in suspension culture. Scale bar = 300 μm (C) Mean ± SD of fold change in cell density for MDA-Tpm2.1 and MDA-EV cells cultured in suspension for 5 and 8 days, measured from three technical replicates Statistical significance was determined using two-way ANOVA followed by Tukey’s multiple comparisons test. (D) Pseudocolor flow cytometry plots of Annexin V and propidium iodide (PI) staining in cells cultured in suspension for 4 days show increased apoptosis in MDA-Tpm2.1 cells. Quadrants reflect populations enriched for live(Q4), early apoptotic(Q3), and late apoptotic(Q2) or necrotic(Q1) cells based on Annexin V and PI signal intensities. (E) Mean ± SEM of total apoptotic cells (%) in attached (1 day) and suspension at 5 h, 1 day, 2 days, 4 days, and 8 days. Data represents two independent biological replicates with 2-3 technical replicate wells per condition.…

• 

  Fig. 2. Transcriptomic profiling of apoptotic and non-apoptotic subpopulations in Tpm2.1-expressing MDA-MB-231 cells under suspension conditions. (A) Schematic of the experimental design. MDA-MB-231 cells stably expressing an empty vector (MDA-EV) or Tpm2.1 (MDA-Tpm2.1) were cultured under adherent or suspension conditions. At each suspension time point (5 h, 1 day, 2 days, and 4 days), MDA-Tpm2.1 cells were separated into apoptotic (Annexin V-positive) and non-apoptotic (Annexin V-negative) subpopulations using Annexin V-conjugated magnetic microbeads, followed by transcriptomic profiling. (B) Spearman correlation heatmap of all RNA-seq samples across time points and conditions. Correlation strength is color-coded, with red indicating strong positive correlation and blue indicating strong negative correlation. The transcriptional profile of apoptotic Tpm2.1-expressing cells at day 4 (Tpm_Apop_4d) is most distinct from all other conditions. (C) Volcano plots showing differentially expressed genes between Tpm_NoAp and Tpm_Apop populations relative to MDA-EV at day 4. Red and green dots represent significantly upregulated and downregulated genes, respectively (adjusted p < 0.05,…

• 

  Fig. 3. KEGG pathway enrichment analyses for MDA-MB-231 cancer cells induced by Tpm2.1 overexpression and suspension growth conditions. (A) KEGG pathway enrichment analysis of upregulated and downregulated genes in MDA-EV, Tpm_NoAp, and Tpm_Apop cells after 5 h and 4 days in suspension, relative to their respective cell populations cultured under attached (rigid) conditions used as controls (MDA-EV, and MDA-Tpm2.1, respectively). Red bars represent upregulated genes. Green bars represent downregulated genes. All pathways have adjusted p < 0.05, |log2FoldChange| > 1. (B,C) Heatmaps of differentially expressed genes from KEGG analysis for the Cell Adhesion Molecules (CAM) pathway (B) and the PI3K-Akt signaling pathway (C). Expression levels are color-coded, with red indicating high expression and green indicating low expression. Heatmaps were generated using an R script, with adjusted p-value (padj) < 0.05 and |log2FoldChange| > 1, applying Pearson correlation and the centroid method for clustering.

• 

  Fig. 4. Change in cell-cell interaction affects anoikis in MDA-Tpm2.1 cells. (A) Phase-contrast images of MDA-EV and MDA-Tpm2.1 cells cultured at three different cell densities for 4 days in suspension. MDA-EV cells are indicated by arrows, MDA-Tpm2.1 cell aggregates are highlighted by circles. Scale bar = 750 μm (B) Mean ± SD of total apoptotic cells (Annexin V+ only, PI+ only, and Annexin V+/PI+; %) in suspension cultures at different cell densities for 4 days. Statistical significance was determined using two-way ANOVA followed by Šídák’s multiple comparisons test. (C,D) Mean ± SD of total apoptotic cells (Annexin V+ only, PI+ only, and Annexin V+/PI+; %) in MDA-EV (C) and MDA-Tpm2.1 (D) cells treated with ICAM-1 inhibitor (ICAM-1-IN-1, 100nM) for 1 day or 4 days under suspension and attached conditions. The ICAM-1 inhibitor was added at the time of seeding. Statistical significance was determined using unpaired t-test followed by Welch’s correction and Holm-Šídák adjustment for multiple comparisons (α = 0.05). (**) for p value < 0.01, (***) for p value < 0.001, (****) for p value <0.0001.

• 

  Fig. 5. Tpm2.1 expression downregulates the Akt and EphA2 pathways in MDA cells under suspension growth conditions. (A) Western blot showing the expression of phospho-Akt (T308) and total Akt in MDA-EV and MDA-Tpm2.1 cells cultured under suspension and attached conditions for 5 days. (B,C) Quantification of phospho-Akt (T308), N=3 (B), and total Akt levels, N=2 (C), in MDA-EV and MDA-Tpm2.1 cells under suspension and attached conditions for 5 days, presented as Mean ± SD. Statistical significance was determined using two-way ANOVA. (D,E) Quantification of total apoptotic cells (%) presented as Mean ± SD for MDA-EV (D) and MDA-Tpm2.1 (E) cells after treatment with Akt inhibitor (5 µM) for 4 days under suspension and attached conditions. The Akt inhibitor was added at the time of seeding (0 h), 1 day, and 2 days after seeding, as indicated on the X-axis of panels D and E. Statistical significance was determined using two-way ANOVA followed by Šídák’s multiple comparisons test. (F) Western blot showing the expression of EphA2 in MDA-EV and MDA-Tpm2.1 cells cultured under suspension and attached conditions for 5 days. (G) Quantification of EphA2 levels in MDA-EV and MDA-Tpm2.1 cells under…

• 

  Fig. 6. Illustrated Working Model. Schematic model depicting the distinct signaling pathways activated in breast cancer cells (MDA-EV) and Tpm2.1-expressing cells (MDA-Tpm2.1), following their separation into non-apoptotic (TpmNoAp) and apoptotic (TpmApop) subpopulations after 5 h and 4 days in suspension. The model highlights subpopulation-specific pathway adaptations and the associated cellular fates under anchorage-independent conditions. Created in https://BioRender.com.