Summary
Cell biologist Michael Overholtzer explains apoptosis, a form of programmed cell death that can lead to cancer when it doesn’t function properly.
The death of one tiny cell might seem like a simple thing. But the process is much more complicated than you would expect.
There are actually several types of cell death, each with its own unique characteristics and processes. One of the most well-studied is called apoptosis.
“Apoptosis is defined by a set of physical, often visible, features that are associated with the demise of an individual cell,” says Memorial Sloan Kettering cell biologist Michael Overholtzer. “It’s probably one of the most common forms of cell death during the development of an organism. It also plays an important role in cancer.”
One purpose of apoptosis is to eliminate cells that contain potentially dangerous mutations. If a cell’s apoptosis function is not working properly, the cell can grow and divide uncontrollably and ultimately create a tumor.
Visible Changes, Molecular Changes
So what do you see if you look at an apoptotic cell under a microscope? “The nucleus looks shrunken or condensed and fragmented into pieces, whereas a normal nucleus is one round oval,” Dr. Overholtzer explains.
“The other obvious feature is that the cells themselves would shrink and begin to bleb,” he adds. “Blebbing means that the cell’s membrane changes, and there are bulging protrusions from the surface of the cell.”
An enzyme called caspase starts the chain reaction of changes that lead to a cell’s death by apoptosis. “Caspase is essentially like molecular scissors,” Dr. Overholtzer says. “In a normal, happy cell, it’s inactive, but once a cell is either put under stress or developmentally programmed to commit suicide, the scissors are activated and start to cut up certain proteins inside the cell, beginning the apoptosis process.”
In cancer cells, however, the scissors may not get the signal to start cutting. “The sensor that recognizes cell damage may not work, and the signal is never sent,” he says. “There are currently strategies under way to develop drugs that would help reactivate the sensor and therefore activate apoptosis.”
One of those sensors is a well-studied protein called p53. When it functions normally, it suppresses the formation of cancer through apoptosis. Mutations in the p53 gene are found in about half of all cancers.
Other Forms of Cell Death
Another common form of cell death is necrosis, which occurs when a cell is injured or ruptured. “Historically we thought that necrosis just happened in response to an injury, for example a burn or a cut,” Dr. Overholtzer says. “In the past five years, however, studies have shown that necrosis is also programmed, but in a completely different way than apoptosis.”
A third well-studied type is called autophagy, in which a cell breaks down and digests itself. Autophagy can occur as a natural part of growth and development, but it can also be a response to certain diseases or stress, such as infection. Similar to apoptosis, autophagy can play a role in cancer, if it doesn’t occur when it should and cells are able to grow out of control.
Dr. Overholtzer’s lab is studying yet another kind of cell death, called entosis, in which one cell engulfs another and kills it. “Entosis is a completely different process,” he says. “The other types of cell death are all forms of suicide, but this is really a murdering event.”
He first discovered entosis as a postdoctoral fellow while studying breast cancer cells under a microscope, when he observed some of the cancer cells entombing neighboring cells. Some of the captured cells escaped unharmed, and some were able to continue dividing inside the host cell, but most eventually died.
Other cases of cells murdering cells that have been observed are different because they result from two different cell types, which are in many cases also distinct genetically, competing against each other. “In this case, cells are killing cells that are identical to themselves,” Dr. Overholtzer says. “We’ve even seen cases where a cell has divided and one daughter cell kills the other one.”
Investigators don’t know yet whether entosis occurs in healthy, normal cells, but it’s something that Dr. Overholtzer and his team are continuing to study.
“Any time you have a new window into how cell death works, particularly a program that you can see going on in a tumor, that’s a real opportunity to dive in and see if you can learn how to manipulate it with the goal of finding new cancer treatment strategies,” he concludes.
Narration
“Apoptosis” is a funny word that is derived from the Latin meaning “to fall off”, like a leaf falls off a tree. And a leaf falls off a tree when it’s dead. And apoptosis refers to a process of what’s called programmed cell death where the cell is actually in a funny kind of way committing suicide. And when this happens, there’s a whole scripted choreography of pathways and proteins within a cell that get activated to actually kill the cell and without making too much of a mess. And this happens normally during development, for instance, in the development of the hand, that normally to begin with, the hand looks very much like a duck paddle foot and the webs between the fingers. Those cells apoptose, giving you the fingers. There are human conditions where that ceases to where apoptosis just does not happen and people are born with web feet. Apoptosis normally happens in cells that have been around in the body long enough that they’re kind of worn out, and so they need to make way for nice, new young cells. When that doesn’t happen, that’s cancer. And so apoptosis can be normal, and in the absence of apoptosis, that can lead to cancer. Too much apoptosis in an otherwise normal human being will result in a number of so-called neurodegenerative diseases where cells die when they’re not supposed to die. And they get messages from some place, most of which we don’t understand, to tell them to die, so in a certain part of the lower part of the brain, that’s what causes Parkinson’s disease. This also characterizes Huntington’s disease, and Alzheimer’s disease, and Lou Gehrig’s disease, and a number of other neurodegenerative diseases.
3.1.1. Phagocytic Clearance and Anti-Inflammatory Signalling
42,43,3CL-1 (fractalkine), which is known to be associated with extracellular vesicles (EVs) released by apoptotic lymphoid cells [
Far from being inert entities, apoptotic cells induce varied responses in their neighbourhood through the active exposure and release of an array of bioactive components (see [ 38 ] (especially Table 3.1), [ 41 44 ] for detailed reviews). Indeed, the secretomes of dying cells that are generated during cancer therapy have important functional effects in facilitating tumour evolution and metastasis [ 45 ]. The most renowned characteristic of an apoptotic cell is its ability to elicit its own phagocytosis by neighbouring tissue cells of various lineages, including dendritic cells, fibroblasts, myocytes, endothelial cells, mesangial cells, Sertoli cells, Paneth cells and most predominantly macrophages, the most readily observed phagocytes of apoptotic cells and the most extensively studied. Indeed, the process of apoptotic-cell engulfment is so swift that free apoptotic cells are rarely observed in situ. Orchestration of the phagocyte’s responses in mediating clearance of apoptotic cells (a process often referred to as efferocytosis) requires transduction of ‘find-me’ and ‘eat-me’ signals generated by the dying cells (reviewed recently [ 43 ]). Examples of the ‘find-me’ signals that attract phagocytes to the locations of apoptosis in mammals include (a) bioactive lipids lysophosphatidylcholine (LPC) [ 46 ] and sphingosine-1-phosphate (S1P) [ 47 ], (b) the classical chemokine CXCL-1 (fractalkine), which is known to be associated with extracellular vesicles (EVs) released by apoptotic lymphoid cells [ 48 ] and (c) nucleotides ATP and UTP, which are released through effector caspase-3-mediated activation of the membrane channel pannexin I [ 49 50 ]. The latter controls transmembrane passage of molecules up to approximately 1kDa through the formation of ion channels in the plasma membrane [ 51 ]. These different classes of chemoattractants all activate chemotaxis of mononuclear phagocytes by binding to specific G protein-coupled receptors. It is noteworthy that the responses of phagocytes to these factors are not limited to migration alone but can include, in addition, stimulation of engulfment mechanisms (for example, through MFG-E8 induction by fractalkine [ 52 ] and polarisation of macrophages from pro-inflammatory to anti-inflammatory states, as well as the promotion of lymphangiogenesis and metastasis in murine tumour models (activation of S1PR1 by S1P [ 53 54 ]). Leukocyte chemotactic control by release of soluble factors from apoptotic cells can also select for the accumulation of mononuclear phagocytes by inhibiting the migration of granulocytes. This is seen in the case of lactoferrin (LTF) which is released during apoptosis and restricts the migration of neutrophils and eosinophils [ 55 56 ]. Although this may represent an important aspect of the anti-inflammatory effect of apoptosis, its role in determining the cellularity of the TME has not been investigated.
vβ3/5, LRP1) engage with PS exposed on apoptotic cells. Indirect interaction with PS is achieved via PS-binding bridging receptors such as Gas6 in the case of MERTK, MFG-E8 for the integrins αvβ3 and αvβ5, while C1q bridges to LRP1. Additional receptors, such as the scavenger receptors CD36 and SR-A, as well as the PRRs CD14 and MBL, recognise apoptotic cells via PS-independent mechanisms (reviewed previously [41,43,
The most renowned ‘eat-me’ signal is the anionic phospholipid, phosphatidylserine (PS), which is actively externalized in the plasma membrane leaflet during apoptosis. This is achieved through the coordinated caspase-dependent (a) inhibition of flippase activity (P4-ATPase ATP11C), which otherwise maintains PS localisation in viable cells to the inner aspect of the plasma membrane leaflet, and (b) activation of scramblase activity (Xkr family member Xkr8), which accelerates the redistribution of PS across the membrane bilayer [ 57 ]. While PS exposure may be necessary for an apoptotic corpse to be engulfed, it is however, insufficient. Additional, ill-defined, components are required, such as carbohydrate changes and binding sites for thrombospondin or other bridging molecules, as well as loss of ‘don’t-eat-me’ signals, such as CD47 and CD31 [ 38 ]. Multiple phagocyte receptors that participate in efferocytosis can either directly (BAI1, TIM4, Stab2, TLT and RAGE) or indirectly (MERTK, α, LRP1) engage with PS exposed on apoptotic cells. Indirect interaction with PS is achieved via PS-binding bridging receptors such as Gas6 in the case of MERTK, MFG-E8 for the integrins αand α, while C1q bridges to LRP1. Additional receptors, such as the scavenger receptors CD36 and SR-A, as well as the PRRs CD14 and MBL, recognise apoptotic cells via PS-independent mechanisms (reviewed previously [ 38 44 ]).
Caenorhabditis elegans
. These have been reviewed elsewhere ([43,
C. elegans
). Either pathway can activate Rac-1 (although the connectivity been GULP1 and Rac activation remains unclear) and this leads to cytoskeletal rearrangement, actin polymerisation and engulfment with subsequent phagosome maturation and lysosomal degradation of phagocytosed cargoes (reviewed recently [2, PGI2 and PAF. In concert, key pro-inflammatory mediators, such as TNFα, IL-1β, IL-8 and IL-12, are downregulated. Details of the molecular mechanisms linking recognition to phagocytosis and inflammation control are emerging. Some receptors, CD14, for example, are mainly involved in tethering of apoptotic cells to phagocytes, whereas others, for example, BAI1 and Stab2, clearly signal Rac-dependent phagocytosis and downstream lysosomal processing. Modification of the aforementioned canonical phagocytosis signals by a process known as LAP (LC3-associated phagocytosis) can speed up lysosomal fusion and degradation and promote anti-inflammatory cytokine production, while abrogation of LAP slows fusion and degradation and promotes pro-inflammatory cytokine release. The details of LAP signaling, including its initiation, are not fully understood, but involve the formation, following receptor-mediated phagocytosis, of a phosphatidylinositol 3-kinase (PI3K) complex comprising at least five proteins, UVRAG, Rubicon, Beclin 1, VPS15 and VPS34. Activation of this PI3K complex results in ligation of LC3 to the lipid membrane of the phagosome and consequent acceleration of lysosomal fusion, cargo degradation and immunosuppressive signaling [
Similar to the death effector machinery of apoptosis, the phagocytosis programme in apoptotic corpse clearance is conserved from worms to mammals, engulfment involving two pathways, originally identified in the nematode worm,. These have been reviewed elsewhere ([ 41 58 ]), each pathway comprising three genes: (a) CED-1, (cell death defective-1) -6 and -7 and (b) CED-2, -5 and -12. Mammalian orthologues of CED-1 are MEGF-10 and LRP1, CED-6 is the engulfment adapter GULP1; CED-7 is represented by ABCA1/7. In the second pathway, CED-2, -5 and -12 are represented in mammals by CrkII, Dock180 and ELMO, the latter two proteins acting as an unconventional two-component guanine nucleotide exchange factor for activating the Rho-family GTPase, Rac-1 (CED-10 in). Either pathway can activate Rac-1 (although the connectivity been GULP1 and Rac activation remains unclear) and this leads to cytoskeletal rearrangement, actin polymerisation and engulfment with subsequent phagosome maturation and lysosomal degradation of phagocytosed cargoes (reviewed recently [ 43 ]). Engulfment of apoptotic cells is typically accompanied by activation of anti-inflammatory responses involving up-regulation of multiple factors, including TGF-β1, IL-10, PGE, PGIand PAF. In concert, key pro-inflammatory mediators, such as TNFα, IL-1β, IL-8 and IL-12, are downregulated. Details of the molecular mechanisms linking recognition to phagocytosis and inflammation control are emerging. Some receptors, CD14, for example, are mainly involved in tethering of apoptotic cells to phagocytes, whereas others, for example, BAI1 and Stab2, clearly signal Rac-dependent phagocytosis and downstream lysosomal processing. Modification of the aforementioned canonical phagocytosis signals by a process known as LAP (LC3-associated phagocytosis) can speed up lysosomal fusion and degradation and promote anti-inflammatory cytokine production, while abrogation of LAP slows fusion and degradation and promotes pro-inflammatory cytokine release. The details of LAP signaling, including its initiation, are not fully understood, but involve the formation, following receptor-mediated phagocytosis, of a phosphatidylinositol 3-kinase (PI3K) complex comprising at least five proteins, UVRAG, Rubicon, Beclin 1, VPS15 and VPS34. Activation of this PI3K complex results in ligation of LC3 to the lipid membrane of the phagosome and consequent acceleration of lysosomal fusion, cargo degradation and immunosuppressive signaling [ 43 ]. It has also been established recently that chloride sensing and flux involving the solute carrier (SLC) proteins SLC12A2 and SLC12A4 play important roles in triggering anti-inflammatory efferocytosis [ 59 ].
Tumour-associated macrophages (TAMs) constitute a significant proportion of the cellular compartment of the TME in diverse malignancies where, in many cases, they are clearly active in clearance of apoptotic cells. As we discuss later, TAM accumulation and pro-oncogenic activation, at least in certain cancers, is closely coupled to tumour growth and angiogenesis. However, little is yet known of the relative contributions of the various find-me, eat-me and anti-inflammatory signaling mechanisms and molecules described above in the pro-oncogenic responses of TAMs to apoptotic tumour cells. Notably, the protein tyrosine kinase MERTK (an indirect PS receptor) is functional in signaling not only phagocytosis of apoptotic cells but also anti-inflammatory/immunosuppressive responses; its inhibition can suppress cancer growth (reviewed in [ 60 ]). It seems likely that phagocyte receptors will orchestrate context-dependent immune responses to apoptotic tumour cells whether dependent on, or independently of, PS; future work will define what receptors are important in specific tumours. An informative example is post-partum breast carcinoma, which tends to present as an aggressive, metastatic disease. In a mouse model that used the involuting mammary fat pad as a breast cancer transplant microenvironment, the critical importance of constitutive apoptosis, MERTK-dependent efferocytosis and TGFβ production were demonstrated [ 7 ].
