Targeting the AKT–EphA2 Axis and Cell–Cell Adhesion Enhances Anoikis Sensitivity in Cancer Cells

Rigidity sensing enables cells to respond to extracellular matrix stiffness and governs survival and apoptotic decisions. Tropomyosin 2.1 (Tpm2.1), a key actin-binding protein, is frequently downregulated in cancer. While re-expressing Tpm2.1 restores rigidity sensing and limits anchorage-independent growth, its ability to fully trigger anoikis, apoptosis induced by loss of matrix attachment, remains unclear. Here, we show that although Tpm2.1 overexpression reduces AKT activation and suppresses survival signaling in suspended breast cancer cells, most cells remain viable without matrix attachment. Time-resolved transcriptomic profiling of apoptotic and non-apoptotic suspended cells revealed compensatory survival programs, including reactivation of PI3K–Akt signaling and upregulation of intercellular adhesion molecules such as ICAM1. Importantly, anoikis resistance can be overcome. Pharmacological inhibition of AKT, EphA2, or ICAM1 in Tpm2.1-expressing cells significantly increases apoptosis under suspension conditions. These findings highlight that while rigidity sensing restoration alone may be insufficient to induce anoikis, it sensitizes cancer cells to targeted therapies, revealing actionable vulnerabilities to eliminate detached tumor cells.