In a recent paper, published in Nature, researchers have discovered that blebbing might be a survival strategy in melanoma cells, and that bleb formation serves as a defence mechanism against anoikis, a type of cellular apoptosis triggered by detachment.
Adhesion or anoikis – cells should stick together
Attachment-dependent survival is essential for cells in multicellular organisms. Cells need to attach to either the extracellular matrix or other cells to generate pro-survival signals. These signals are produced by adhesive structures, such as focal adhesions and adherens junctions, which control the concentration of signalling factors, protein scaffolds, and effector proteins. If cells are deprived of attachment, they undergo anoikis, which triggers cellular apoptosis.
When cells are detached, they become rounded and exhibit dynamic surface blebs. These are cellular protrusions, and can be found in healthy cells. However, detached non-malignant cells only maintain dynamic blebbing for a couple of hours, and undergo anoikis if attachment is not re-established. Cancer cells, on the other hand, are highly anoikis resistant, and acquiring molecular strategies that confer anoikis resistance is a critical step in oncogenesis. One example of this is how cancer cells are able to indefinitely sustain rounded blebby morphologies in no- and low-attachment environments, often adopting this amoeboid-associated phenotype both in vitro and in vivo.
Bleb formation has long been associated with metastatic melanoma and other aggressive cancers. Recent studies have shown that blebbiness is associated with increased metastatic potential in melanoma and prostate cancer.Therefore blebbiness might be an indicator of disease aggressiveness. Although the correlation between blebby morphologies and metastasis has largely been attributed to the role of blebs in cell motility, recent work has suggested that blebbiness might also convey a survival advantage. Signalling factors associated with cell survival appear to be concentrated in the vicinity of melanoma blebs, including KRAS and active PI3K. The PI3K pathway has been found to be dysregulated in almost all human cancers, and recent studies have shown that inhibition of blebbing causes a sharp reduction in local PI3K activity, increasing the possibility that blebbing directly enables this signalling.
The blebbier, the better
In an effort to understand how cancer cells sustain blebby morphologies and how they are associated with metastasis and survival, the research team at UT Southwestern Medical Center decided to investigate blebbing as a potential contributor to anoikis resistance in melanoma. The researchers found that non-malignant mammalian cells that sustain prolonged blebbing after detachment exhibit increased resistance to anoikis. This resistance was dependent on the formation of bleb-associated septin structures, and inhibition of either bleb formation or septin assembly restored sensitivity to anoikis. The study suggests that cancer cells and non-malignant cells use similar bleb- and septin-dependent mechanisms to evade anoikis.
The researchers first inhibited bleb formation in melanoma cells using wheat germ agglutinin (WGA) to minimize cell attachment over a 24-hour period. They found that bleb inhibition resulted in increased cell death in detached cells but not adhered cells. Septins are proteins involved in cell division and membrane remodelling, and their expression has been shown to be altered in cancer cells. Septins are also the only known eukaryotic proteins that detect the positive micrometer-scale membrane curvature that blebbing creates. The researchers confirmed that septins are primarily found near blebby cell surface regions, and septin structures were enriched at bleb edges. They found that “pulses” of septin accumulation occur at the curvy bases of blebs and fade quickly once the bleb is resolved.
They then investigated whether cortical septin structures are involved in the acquisition of bleb-dependent anoikis resistance in cancer cells. Live-cell imaging showed that poorly adhered cancer cells possess extensive cortical septin structures that are spatially associated with blebs. In contrast, unperturbed cancer cells, which had shown bleb-independent anoikis resistance, exhibited no septin enrichment at the cortex despite robust bleb formation. The researchers also used the forchlorfenuron (FCF) septin inhibitor, and inhibiting cortical septin structures greatly disrupted anoikis resistance for poorly adhered cancer cells, while having no effect on unperturbed cancer cells. They then investigated how septins amplify these pro-survival signals by investigating which proteins interacted with septins, and found that they scaffold the oncogene NRAS. These findings suggest that the mechanism allowing blebs to confer anoikis resistance depends on bleb-adjacent cortical septin structures.
Popping the blebbles – a new target for cancer treatment?
Altogether, these findings suggest that blebbing is a survival strategy in melanoma cells, and bleb formation serves as a defence mechanism against anoikis. It highlights the importance of elucidating the underlying cellular mechanisms behind cell-death resistance in cancer cells. Future research in this area could focus on identifying other proteins or signalling pathways involved in the formation and regulation of septin structures and blebbing, as well as investigating the potential of targeting these mechanisms for cancer therapy.
