Research report: PIK3CA silencing restores anoikis sensitivity in endothelial cells

Silencing the cancer-associated gene PIK3CA restored apoptosis sensitivity and reversed several aggressive traits in an in vitro model of anoikis-resistant endothelial cells, according to a study published in Molecular Biology Reports. The researchers used microRNA interference to reduce expression of the gene, which encodes the p110α catalytic subunit of phosphoinositide 3-kinase, or PI3K. In engineered rabbit aortic endothelial cells, loss of PIK3CA was associated with reduced survival and colony formation after adhesion was blocked, lower invasion, slower proliferation, changes in cell shape and actin organization, and increased activation of apoptotic pathways.

The findings identify PIK3CA as an important molecular dependency in the laboratory model, but they do not establish a cancer treatment or a clinical benefit in people. Anoikis is a form of apoptosis triggered when adherent cells lose contact with neighboring cells or the extracellular matrix. The process helps prevent detached cells from surviving inappropriately, while anoikis-resistant cells can persist without anchorage.

In tumors, that resistance is associated with a more aggressive phenotype and with the ability of cells to survive detachment, enter the circulation and potentially seed distant tissues. The study focused on the relationship between PI3K signaling and this phenotype in endothelial cells, a complementary experimental system to the tumor models more commonly used to investigate anchorage-independent survival. PIK3CA is especially notable because it is among the most frequently mutated oncogenes in human cancers and because PI3K/AKT signaling regulates cell survival, proliferation, differentiation, metabolism and cell-cycle progression.

The investigators began with wild-type rabbit aortic endothelial cells, EJ-ras-transfected endothelial cells and an anoikis-resistant line designated Adh1-EC, which had been established through successive cycles of adhesion blockade. They generated two additional Adh1-EC-derived lines carrying a plasmid expressing a microRNA that targets PIK3CA, along with a control line transfected with a non-targeting microRNA. The vector also carried an emerald green fluorescent protein reporter, and insertion of the microRNA sequence was confirmed by Sanger sequencing.

Knockdown was assessed at both RNA and protein levels through real-time reverse-transcription PCR, western blotting and immunofluorescence. The two silenced clones showed reductions in PI3K p110α protein of 56% and 83% relative to the parental anoikis-resistant cells. β-actin and GAPDH expression did not differ significantly between silenced cells and controls, which the investigators used as evidence that the manipulation was selective. Morphological analysis indicated that the silenced cells had shifted toward the appearance of wild-type endothelial cells.

Under scanning electron microscopy, the anoikis-resistant and EJ-ras-transfected lines had fewer surface protrusions and less spreading, whereas PIK3CA-silenced cells were more broadly distributed across the substrate and showed structures resembling the filopodia and lamellipodia of control endothelial cells. Fluorescence imaging also indicated a more regular arrangement of filamentous actin in the silenced cells, with fibers distributed more widely than in parental Adh1-EC cells. In adhesion assays, knockdown significantly increased attachment to fibronectin at the concentrations tested.

Adhesion to collagen IV and laminin, however, did not change significantly. The selective response indicates that not every form of extracellular-matrix attachment in this model depends on PIK3CA; the investigators noted that other receptors or compensatory pathways may support attachment to those substrates. Functional changes followed PIK3CA silencing under both anchorage-independent and adherent conditions.

A resazurin-based viability assay showed significantly lower metabolic activity among the PIK3CA-silenced cells after 96 hours of adhesion blockade, consistent with renewed sensitivity to anoikis. The same cells had a reduced ability to form colonies when plated on agarose, maintained in suspension and subsequently returned to adherent culture. Their proliferation rate under standard adherent conditions also fell, with the strongest reported effect at 96 hours.

In Transwell assays using a matrix containing entactin, collagen IV and laminin, the silenced cells migrated through the 8-micrometer-pore membrane at a significantly lower rate than the parental anoikis-resistant line. These measurements collectively indicate that PIK3CA supports survival without anchorage and maintains several behaviors associated with a malignant phenotype in this experimental system. Apoptosis measurements provided a mechanistic counterpart to those functional observations.

Flow-cytometric analysis with annexin V and propidium iodide showed a higher apoptotic rate in the PIK3CA-silenced cells than in parental Adh1-EC cells. At the transcriptional level, the silenced cells had lower BCL2 messenger RNA, higher BAD messenger RNA and higher BAX messenger RNA. BCL2 encodes an anti-apoptotic protein, whereas BAD and BAX encode pro-apoptotic members of the Bcl-2 family.

No significant differences were detected in the measured BCL2 or BAD protein abundance, so the changes in messenger RNA did not translate into corresponding measured changes at the protein level for those two markers. The study also detected increased CASP3 messenger RNA and caspase-3 protein in the silenced cells, together with greater caspase-3/7 and caspase-8 activity. Because caspase-8 activity is associated with initiation of the extrinsic apoptotic pathway and caspase-3/7 activity reflects the execution of apoptosis through intrinsic and extrinsic signals, the results support involvement of both routes after PIK3CA knockdown.

The authors note that activated AKT, a downstream PI3K effector, can promote survival by phosphorylating and inactivating proteins such as BAD and caspase-9 and by increasing transcription of anti-apoptotic Bcl-2 family members. In the present model, reducing PIK3CA was followed by a molecular shift compatible with reactivation of apoptosis, although the downstream links between PIK3CA, cytoskeletal organization, extracellular-matrix remodeling and cell death were not fully resolved. The lower resazurin signal after adhesion blockade was therefore interpreted mainly as increased apoptosis and reduced proliferation, although the investigators cautioned that diminished metabolic activity could also contribute.

The work links one PI3K component to a coordinated set of cellular behaviors rather than a single isolated effect. PIK3CA silencing altered cytoskeletal organization, reduced invasion and increased attachment to fibronectin while restoring anoikis sensitivity. Its effects were detectable across complementary assays: microscopy documented structural changes, adhesion plates measured attachment to defined matrix components, Transwells quantified migration, agarose culture tested survival without anchorage, colony assays assessed proliferative capacity after detachment, and molecular measurements tracked apoptotic signaling.

This breadth supports the conclusion that the phenotype was not merely a change in one assay. At the same time, the experiments were performed in one engineered endothelial cell model, and the researchers did not test animals or people. It remains unknown whether the same dependency and downstream responses occur in different tumor types, primary endothelial cells or other anoikis-resistant models.

For cancer biology, the results point to a potentially important distinction between merely observing elevated PI3K/AKT signaling in malignant cells and functionally showing that PIK3CA is required to sustain their phenotype. The intervention was genetic silencing in cultured cells, not a drug treatment, and it produced broad changes in survival, behavior and cell death rather than demonstrating that PIK3CA inhibition is safe or effective in patients. The study therefore does not support clinical recommendations.

Its more immediate significance is experimental: PIK3CA appears to coordinate anchorage-independent survival with proliferation, invasion and adhesion in Adh1-EC cells. Additional studies would need to test whether that relationship is conserved across independent endothelial and tumor models and to identify the signaling routes connecting PIK3CA to integrin-mediated adhesion, extracellular-matrix remodeling and the apoptotic machinery. Those questions will determine whether the silenced phenotype offers a useful basis for wider mechanistic studies or a strategically targeted intervention.

Subject of Research: PIK3CA-dependent anoikis resistance in an in vitro endothelial cell model

Article Title: PIK3CA silencing restores anoikis sensitivity in endothelial cells

Article References: Pereira, D. M. C., de Sousa Mesquita, A. P., da Silva Cruz, A., Donatti, L., Nader, H. B., & Lopes, C. C. (2026). PIK3CA silencing restores anoikis sensitivity in endothelial cells. Molecular Biology Reports, 53(1), Article 1500. https://doi.org/10.1007/s11033-026-12680-3

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12680-3

Keywords: PIK3CA, anoikis, PI3K signaling, endothelial cells, apoptosis, cancer biology, microRNA interference, cell invasion, extracellular matrix, rabbit aortic cells, silencing, restores