Cancer Cell Biol

Question 1

What is an exosome?

Answer 1

Exosomes are tiny vesicles that are enriched in
nucleic acids and proteins and released from
cells.

Question 2

How might exosomes play a role in cancer biology?

Answer 2

Originally considered to have no biologic
significance, these nano-sized blebs are now considered
to be mini-maps of their cells of origin,
with physiological and pathologic relevance. In
cancer, they have been implicated in the muddling
of cell-to-cell communication and in the
transfer of “undesirable” information from one
cell to another. Consequences include stimulating
the proliferation, motility, and invasive properties
of the recipient cell, transferring drug resistance,
inducing the formation of endothelial
tubules (e.g., in angiogenesis), and attracting
cancer cells to secondary sites within living organisms. MicroRNA content seems to be particularly instrumental.

Question 3

What is microRNA?

Answer 3

MiRNAs are short, double-stranded
RNA fragments that are generated from precursor
miRNAs (pre-miRNAs). They do not encode
proteins but, rather, regulate the levels of expression
of specific sets of messenger RNAs
(mRNAs), and therefore their protein products,
by mechanisms that include binding to these
mRNAs and targeting them for degradation.
This binding-and-degradation process requires
the pre-miRNA to be incorporated into a multiprotein
complex called the RNA-induced silencing
complex–loading complex. Within this complex,
pre-miRNAs mature into miRNAs by means
of their interaction with two proteins: an enzyme
called Dicer and the transactivating response
RNA binding protein (TRBP). Finally, a protein
called argonaute 2 (AGO2) binds to miRNA and
guides it to its target complementary mRNA.

Question 4

Melo et al: evidence for role of exosomes in breast cancer?

Answer 4

Starting with exosomes from breast-cancer cell lines and those from nontumorigenic breastcell
lines (human, MCF-10A; mouse, NMuMG),
Melo et al.1 found that the exosomes derived
from the cancer cell lines but not those from the
nontumorigenic breast cells were enriched in
miRNAs and that the exosomes from cancer
cells could convert pre-miRNAs into mature
miRNAs. They cultured exosomes from both
types of cells separately for 3 days and monitored
the conversion of six pre-miRNAs into miRNAs
(including two specific miRNAs that are known
to be relevant to breast-cancer biology, miR-10b
and miR-21). In exosomes derived from the
breast-cancer cell lines, the ratio of miRNA to
pre-miRNA increased with time, indicating active
miRNA formation. The investigators did not
detect a change in this ratio in the exosomes
derived from nontumorigenic breast cells. Next,
the group introduced synthetic pre-miRNAs into
exosomes from the cancer cells; these were converted
to miRNAs over the same time frame.
Consistent with these findings was the detection
of Dicer, TRBP, and AGO2 in exosomes that were
derived from breast-cancer cells only. To test the effect of exosomal contents on
normal cells, the authors exposed MCF-10A cells
to exosomes that were derived from the breastcancer
cell line MDA-MB-231. Exposure of these
normal cells to the exosomes that were derived
from the cancer-cell line and cultured over a
period of 3 days increased cell survival and proliferation.
This effect was accompanied by decreased
expression of the tumor-suppressor protein
PTEN and the transcription factor HOXD10,
which suppresses the expression of genes that
promote invasion, migration, and tumor progression.
The investigators then found that the nontumorigenic
cells, when coinjected with exosomes
from the cancer cells into mice, formed
tumors — unless Dicer activity was
blocked, which suggests that Dicer is critical to the transformation of normal cells into tumor
cells on exposure to exosomes.
Melo and colleagues also found that serum
specimens from patients with cancer had more
exosomes than did those from healthy controls.
They also observed that the same six pre-miRNAs,
when cultured, matured to miRNAs in the exosomes
from patients but not in those from
healthy donors. The exosomes from 5 of 11 of
these patients, when injected with the nontumorigenic
breast epithelial (MCF-10A) cells, induced
tumor formation in mice; those from 8 healthy
donors did not.

Question 5

What did Le et al demonstrate about the potential role of exosomes and ectosomes in breast cancer metastasis?

Answer 5

Metastasis to secondary organs is the major
cause of death from breast cancer and often
involves an epithelial cell–to–mesenchymal cell
transformation and subsequent reversion to epithelium,
a process that is regulated by the miR-200
family of miRNAs. Le and colleagues found that
exosomes and larger vesicles (termed ectosomes) can transfer miR-200s from highly metastatic
cells to poorly metastatic cells and thereby increase
the metastatic potential of the poorly metastatic
cells.2 To begin, they used mouse triplenegative
breast-cancer cells. Mouse triple-negative
breast-cancer cells that were poorly metastatic
were exposed for 3 days to either their own extracellular
vesicles or to those derived from the
highly metastatic mouse triple-negative breastcancer
cell line, 4TE1. When injected into the tail
vein of mice, the cells that were preincubated
with the extracellular vesicles from highly metastatic
cells formed substantially more lung metastases
than did the cells that were preincubated
with their own extracellular vesicles. This
finding was maintained except when miR-200
family members were blocked, in which case
there were fewer and smaller lung metastases.
Le et al. then carried out similar experiments,
and obtained similar results, with human breastcancer
cell lines.

Question 6

What evidence is there that stromal exosomes can influence cancer growth? Role of metalloproteinases?

Answer 6

It appears that exosomes in the context of the
stromal microenvironment also exert influence on
tumor behavior. Boelens and colleagues3 found
that exposure to stromal exosomes expanded a
subpopulation of breast-cancer cells that are resistant
to therapy and can initiate tumor formation.
Shimoda and colleagues4 found that tissue
inhibitors of metalloproteinases (TIMPs) guard
against the release of tumor-promoting exosomes
by the stroma: a depletion of TIMPs resulted in
cancer-associated fibroblast-like cells. Squamouscell
carcinomas of the head and neck are a
source of TIMP-less fibroblasts; exosomes derived
from such fibroblasts enhance the motility
of breast-cancer cells.

Question 7

What are the 6 hallmarks of cancer proposed by Hanahan and Weinberg in their origin Cell paper (2000)?

Answer 7

We suggest that the vast catalog of cancer cell genotypes is a manifestation of six essential alterations in cell physiology that collectively dictate malignant growth (Figure 1): self-sufficiency in growth signals, insensitivity to growth-inhibitory (antigrowth) signals, evasion of programmed cell death (apoptosis), limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis.

Hallmarks of Cancer:
SARCOMA
S – Self-sufficiency of growth signaling
A – Apoptosis evasion
R – Resistance to anti-growth factor signaling
CO – Continuous replication (Immortality)
M – Metastasis
A – Angiogenesis

Question 8

Give three modes of growth signal transduction.

Answer 8

Normal cells require mitogenic growth signals (GS) before they can move from a quiescent state into an active
proliferative state. These signals are transmitted into the
cell by transmembrane receptors that bind distinctive
classes of signaling molecules: diffusible growth factors,
extracellular matrix components, and cell-to-cell
adhesion/interaction molecules.

Question 9

Hallmarks of cancer: Acquired GS autonomy. Name three common molecular strategies for achieving GS autonomy.

Answer 9

Acquired GS autonomy was the first of the six capabilities
to be clearly defined by cancer researchers, in large
part because of the prevalence of dominant oncogenes
that have been found to modulate it. Three common
molecular strategies for achieving autonomy are evident,
involving alternation of:
1. extracellular growth signals
2. transcellular transducers of those signals
3. intracellular circuits that translate those signals into action.

Question 10

Hallmarks of cancer: Acquired GS autonomy. Name three common molecular strategies for achieving GS autonomy. Give examples of how cancer cells can manipulate extracellular growth signals to become GS autonomous.

Answer 10

Three common molecular strategies for achieving autonomy are evident, involving alternation of:

1. extracellular growth signals
2. transcellular transducers of those signals
3. intracellular circuits that translate those signals into action.

Examples:
1. Manipulation of extracellular growth signals: While most soluble mitogenic growth factors (GFs) are made by one cell type in order to stimulate proliferation of another—the process of heterotypic signaling—many cancer cells acquire the ability to synthesize GFs to which they are responsive, creating a positive feedback signaling loop often termed autocrine stimulation (Fedi et al., 1997). Clearly, the manufacture of a GF by a cancer cell obviates dependence on GFs from other cells within the tissue. The production of PDGF (platelet-derived growth factor) and TGF-alpha by glioblastomas and sarcomas, respectively, are two illustrative examples.

Question 11

Hallmarks of cancer: Acquired GS autonomy. Name three common molecular strategies for achieving GS autonomy. Give examples of how cancer cells can manipulate transcellular transducers of GS to become GS autonomous.

Answer 11

Three common molecular strategies for achieving autonomy are evident, involving alternation of:

1. extracellular growth signals
2. transcellular transducers of those signals
3. intracellular circuits that translate those signals into action.

Examples:
2. Manipulation of transducers of growth signals:
i) Growth factor receptor over-expression may enable the cancer cell to become hyper-responsive to ambient levels
of GF that normally would not trigger proliferation. For example, the epidermal GF receptor (EGF-R/erbB) is upregulated in stomach, brain, and breast tumors, while the HER2/neu receptor is overexpressed in stomach and mammary carcinomas. Additionally, gross overexpression of GF receptors can elicit ligand-independent signaling. ii) Structural alteration of receptors: Ligand-independent
signaling can also be achieved through structural
alteration of receptors; for example, truncated versions
of the EGF receptor lacking much of its cytoplasmic
domain fire constitutively.
iii) Cancer cells can also switch the types of extracellular
matrix receptors (integrins) they express, favoring ones
that transmit progrowth signals. These bifunctional,
heterodimeric cell surface receptors physically link cells
to extracellular superstructures known as the extracellular matrix (ECM). Successful binding to specific moieties of the ECM enables the integrin receptors to transduce signals into the cytoplasm that influence cell behavior, ranging from quiescence in normal tissue to motility, resistance to apoptosis, and entrance into the active cell cycle. Conversely, the failure of integrins to forge these extracellular links can impair cell motility, induce apoptosis, or cause cell cycle arrest. Both ligand-activated GF receptors and progrowth integrins engaged to extracellular matrix components can activate the SOS-Ras-Raf-MAP kinase pathway.

Question 12

Hallmarks of cancer: Acquired GS autonomy. Name three common molecular strategies for achieving GS autonomy. Give examples of how cancer cells can manipulate intracellular circuits that translate GS into action.

Answer 12

Three common molecular strategies for achieving autonomy are evident, involving alternation of:

1. extracellular growth signals
2. transcellular transducers of those signals
3. intracellular circuits that translate those signals into action.

Examples:
3. Alterations in components of the downstream
cytoplasmic circuitry that receives and processes
the signals emitted by ligand-activated GF
receptors and integrins. The SOS-Ras-Raf-MAPK cascade
plays a central role here. In about 25% of human tumors, Ras proteins are present in structurally altered
forms that enable them to release a flux of mitogenic
signals into cells, without ongoing stimulation by their
normal upstream regulators.
- We suspect that growth signaling pathways suffer
deregulation in all human tumors. Although this point
is hard to prove rigorously at present, the clues are
abundant. For example, in the best studied of tumors—human colon carcinomas—about half of the tumors bear mutant ras oncogenes. We suggest that the remaining colonic tumors carry defects in other components of the growth signaling pathways that phenocopy ras oncogene activation.
- The SOS-Ras-Raf-MAP kinase mitogenic cascade is linked via a variety of cross-talking connections with other pathways; these cross connections enable extracellular signals to elicit multiple cell biological effects. For example, the direct interaction of the Ras protein with the survival-promoting PI3 kinase enables growth signals to concurrently evoke survival signals within the cell.

Question 13

Hallmarks of cancer: Acquired GS autonomy. Name three common molecular strategies for achieving GS autonomy. Discuss an addition mechanism of cancer cell growth regulation/deregulation.

Answer 13

While acquisition of growth signaling autonomy by
cancer cells is conceptually satisfying, it is also too
simplistic. We have traditionally explored tumor growth
by focusing our experimental attentions on the genetically deranged cancer cells. It is, however, increasingly apparent that the growth deregulation
within a tumor can only be explained once we
understand the contributions of the ancillary cells present in a tumor—the apparently normal bystanders such as fibroblasts and endothelial cells—which must play key roles in driving tumor cell proliferation. Within normal tissue, cells are largely instructed to grow by their neighbors (paracrine signals) or via systemic (endocrine) signals. Cell-to-cell growth signaling is likely to operate in the vast majority of human tumors as well; virtually all are composed of several distinct cell types that appear to communicate via heterotypic signaling. Successful tumor cells are those that have acquired the ability to co-opt their normal neighbors by inducing them to release abundant fluxes of growth-stimulating signals. Indeed, in some tumors, these cooperating cells may eventually depart from normalcy, coevolving with their malignant neighbors in order to sustain the growth of the latter. Further, inflammatory cells attracted to sites
of neoplasia may promote (rather than eliminate) cancer
cells, another example of normal cells conscripted to enhance tumor growth potential, another means to acquire necessary capabilities.

Question 14

PD-1 Blockade in Tumors

with Mismatch-Repair Deficiency, Le et al (Johns Hopkins), NEJM, June 2015. Main findings/significance.

Answer 14

BACKGROUND
Somatic mutations have the potential to encode “non-self” immunogenic antigens. We hypothesized that tumors with a large number of somatic mutations due to
mismatch-repair defects may be susceptible to immune checkpoint blockade.
METHODS
We conducted a phase 2 study to evaluate the clinical activity of pembrolizumab, an anti–programmed death 1 immune checkpoint inhibitor, in 41 patients with progressive metastatic carcinoma with or without mismatch-repair deficiency. Pembrolizumab was administered intravenously at a dose of 10 mg per kilogram of body weight every 14 days in patients with mismatch repair–deficient colorectal cancers, patients with mismatch repair–proficient colorectal cancers, and patients with mismatch repair–deficient cancers that were not colorectal. The coprimary end points were the immune-related objective response rate and the 20-week immune-related progression-free survival rate.
RESULTS
The immune-related objective response rate and immune-related progression-free survival rate were 40% (4 of 10 patients) and 78% (7 of 9 patients), respectively, for mismatch repair–deficient colorectal cancers and 0% (0 of 18 patients) and 11% (2 of 18 patients) for mismatch repair–proficient colorectal cancers. The median progression-free survival and overall survival were not reached in the cohort with mismatch repair–deficient colorectal cancer but were 2.2 and 5.0 months, respectively, in the cohort with mismatch repair–proficient colorectal cancer (hazard ratio for disease
progression or death, 0.10 [P

Question 15

What is pembrolizumab?

Answer 15

Pembrolizumab is a humanized monoclonal anti–PD-1 antibody of the IgG4 kappa isotype that blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2

Question 16

How can mismatch repair status be evaluated?

Answer 16

Analysis of Mismatch-Repair Status: Tumors with genetic defects in mismatch-repair pathways are known to harbor hundreds to thousands of somatic mutations, especially in regions of repetitive DNA known as microsatellites. The accumulation of mutations in these regions of the genome is termed microsatellite instability. Mismatch-repair status was assessed in tumors with the use of the MSI Analysis System (Promega), through the evaluation of
selected microsatellite sequences that are particularly prone to copying errors when mismatch repair is compromise.

Question 17

Describe how you might estimate the total number of

mutation-associated neoantigens in a patient’s tumor

Answer 17

Primary tumor samples and matched normal peripheral-blood specimens were obtained from a subgroup of patients with mismatch repair– deficient carcinomas and a subgroup with mismatch repair–proficient carcinomas, for whom sufficient tumor tissue was available for exome sequencing and HLA haplotyping. To assess the potential for mutant peptide binding, somatic exome data combined with each individual patient’s major histocompatibility complex (MHC) class I HLA haplotype were applied to an epitope prediction algorithm. This algorithm provided an estimate of the total number of mutation-associated neoantigens in each tumor.
See Le, et al, 2015.

Question 18

Hallmarks of Cancer. Acquired Capability: Insensitivity
to Antigrowth Signals.
Describe two main categories of antigrowth signal.

Answer 18

1. Soluble growth inhibitors.
2. Immobilized inhibitors embedded in extracellular matrix and on the surface of neighbouring cells.

Within a normal tissue, multiple antiproliferative signals
operate to maintain cellular quiescence and tissue homeostasis; these signals include both soluble growth
inhibitors and immobilized inhibitors embedded in the
extracellular matrix and on the surfaces of nearby cells.
These growth-inhibitory signals, like their positively acting counterparts, are received by transmembrane cell
surface receptors coupled to intracellular signaling circuits.

Question 19

Hallmarks of Cancer. Acquired Capability: Insensitivity

to Antigrowth Signals. Describe two distinct mechanisms by which antigrowth signals can block cell proliferation.

Answer 19

1. Force cells into G0.
2. Force cells into postmitotic differentiated state.

Antigrowth signals can block proliferation by two distinct
mechanisms. Cells may be forced out of the active proliferative cycle into the quiescent (G0) state from which they may reemerge on some future occasion when extracellular signals permit. Alternatively, cells may be induced to permanently relinquish their proliferative potential by being induced to enter into postmitotic states, usually associated with acquisition of specific
differentiation-associated traits.

Question 20

Hallmarks of Cancer. Acquired Capability: Insensitivity

to Antigrowth Signals. Which 3 proteins are central to processing most antiproliferative signals?

Answer 20

The retinoblastoma protein (pRb) and its two relatives, p107 and p130.

Much of the circuitry that enables normal cells to respond to antigrowth signals is associated with the cell cycle clock, specifically the components governing the transit of the cell through the G1 phase of its growth cycle. Cells monitor their external environment during this period and, on the basis of sensed signals, decide whether to proliferate, to be quiescent, or to enter into a postmitotic state. At the molecular level, many and perhaps all antiproliferative signals are funneled through the retinoblastoma protein (pRb) and its two relatives, p107 and p130.

Question 21

Hallmarks of Cancer. Acquired Capability: Insensitivity
to Antigrowth Signals.
How does hypophosphorylated pRb block cell proliferation?

Answer 21

When in a hypophosphorylated state, pRb blocks proliferation by sequestering and altering the function of E2F transcription factors that control the expression of banks of genes essential for progression from G1 into S phase.

Question 22

Hallmarks of Cancer. Acquired Capability: Insensitivity

to Antigrowth Signals. What is TGF-beta? What effect does TGF-beta have on pRb and hence on cell cycle progression?

Answer 22

TGF-beta is a soluble signalling molecule that acts as an antigrowth factor. TGF-beta acts in a number of ways to prevent the phosphorylation that inactivates pRb; in this fashion, TGF-beta blocks advance through G1.

Question 23

Hallmarks of Cancer. Acquired Capability: Insensitivity
to Antigrowth Signals.
Name three cell cycle regulatory proteins whose expression TGF-beta can influence.

Answer 23

TGF-beta

1. Suppresses c-myc expression
2. Induces synthesis of p15INK4b and p21

In some cell types, TGF-beta suppresses expression
of the c-myc gene, which regulates the G1 cell cycle
machinery.
More directly, TGF-beta causes synthesis of the p15INK4B and p21 proteins, which block the cyclin:CDK complexes responsible for pRb phosphorylation

Question 24

Hallmarks of Cancer. Acquired Capability: Insensitivity

to Antigrowth Signals. What role do p15INK4B and p21 proteins play in cell cycle progression?

Answer 24

p15INK4B and p21 proteins block the cyclin:CDK complexes responsible for pRb phosphorylation. Hence they keep pRb in its growth-inhibitory hypophosphorylated state -> sequestration of E2F transcription factors.

Question 25

Hallmarks of Cancer. Acquired Capability: Insensitivity

to Antigrowth Signals. What is Smad4 and how does it participate in antigrowth signalling?

Answer 25

The cytoplasmic Smad4 is a cytoplasmic protein that transduces signals from ligand-activated TGF-beta receptors to downstream targets.

Question 26

Hallmarks of Cancer. Acquired Capability: Insensitivity

to Antigrowth Signals. Describe 8 ways in which the pRb signaling circuit can be disrupted in tumours.

Answer 26

The pRb signaling circuit, as governed by TGF-beta and
other extrinsic factors, can be disrupted in a variety of
ways in different types of human tumors.
1. Down-regulation of TGF-beta receptors
2. Mutant, dysfunctional TGF-beta receptors
3. Deletion of the Smad4 gene and hence loss of signal transduction between ligand-actived TGF-beta receptors and downstream targets.
4. Deletion of the locus encoding p15INK4B
5. Insensitivity of CDK4 to the inhibitory actions of p15INK4B due to mutations that create amino acid substitutions in its INK4A/B-interacting domain; the resulting cyclin D:CDK4 complexes are then given a free hand to inactivate pRb by hyperphosphorylation
6. Loss of pRb through mutation of its gene
7. pRb sequestration by viral oncoproteins; this occurs in certain DNA virus-induced tumors, notably cervical carcinomas, in which pRb function is eliminated through sequestration by the E7 oncoprotein of HPV.
8. Cancer cells can also turn off expression of integrins and other cell adhesion molecules that send antigrowth signals, favoring instead those that convey progrowth signals; these adherence-based antigrowth signals likely impinge on the pRb circuit as well.

The bottom line is that the antigrowth circuit converging onto Rb and the cell division cycle is, one way or another, disrupted in a majority of human cancers, defining the concept and a purpose of tumor suppressor loss in cancer.

Question 27

Hallmarks of Cancer. Acquired Capability: Insensitivity
to Antigrowth Signals. What does the c-myc oncogene encode? How do Myc, Max and Mad interact to influence cell differentiation?

Answer 27

The c-myc oncogene encodes a transcription factor. During normal development, the growth-stimulating action of Myc, in association with another factor, Max, can be supplanted by alternative complexes of Max with a group of Mad transcription factors; the Mad–Max complexes elicit differentiation-inducing signals.

Question 28

Hallmarks of Cancer. Acquired Capability: Insensitivity

to Antigrowth Signals. Give 3 examples of ways in which tumour cells avoid terminal differentiation?

Answer 28

Cell proliferation depends on more than an avoidance of cytostatic antigrowth signals. Our tissues also constrain cell multiplication by instructing cells to enter irreversibly into postmitotic, differentiated states, using diverse mechanisms that are incompletely understood; it is apparent that tumor cells use various strategies to avoid this terminal differentiation.

1. Overexpression of the c-Myc oncoprotein, as is seen in many tumors, can shift the balance to favor Myc–Max complexes, thereby impairing differentiation and promoting growth.
2. During human colon carcinogenesis, inactivation of the APC/beta-catenin pathway serves to block the egress of enterocytes in the colonic crypts into a differentiated, postmitotic state.
3. Analogously, during the generation of avian erythroblastosis, the erbA oncogene acts to prevent irreversible erythrocyte differentiation

Question 29

Overview of the roles of TGF-beta in oncogenesis.

Answer 29

1. Deregulated TGF-beta signalling cascades in cancer cells confer resistance to antigrowth signalling.
2. Increased production of TGF-beta by cancer cells and surrounding stromal cells fails to suppress cancer growth, but can promote immunosuppression and angiogenesis due to effects on surrounding microenbironment.

In normal cells, TGF-β, acting through its signaling pathway, stops the cell cycle at the G1 stage to stop proliferation, induce differentiation, or promote apoptosis. In many cancer cells, parts of the TGF-β signaling pathway are mutated, and TGF-β no longer controls the cell. These cancer cells proliferate. The surrounding stromal cells (fibroblasts) also proliferate. Both cells increase their production of TGF-β. This TGF-β acts on the surrounding stromal cells, immune cells, endothelial and smooth-muscle cells. It causes immunosuppression and angiogenesis, which makes the cancer more invasive. TGF-β also converts effector T-cells, which normally attack cancer with an inflammatory (immune) reaction, into regulatory (suppressor) T-cells, which turn off the inflammatory reaction.

Question 30

Role of TGF-beta in immune regulation?

Answer 30

TGF-β is believed to be important in regulation of the immune system by Foxp3+ Regulatory T cell and the differentiation of both Foxp3+ Regulatory T cell and of Th17 cells from CD4+ T cells. TGF-β appears to block the activation of lymphocytes and monocyte derived phagocytes.

Question 31

what is a teleology?

Answer 31

Teleology, (from Greek telos, “end,” and logos, “reason”) = explanation by reference to some purpose, end, goal, or function. For example, a teleological explanation of why forks have prongs is that this design helps humans eat certain foods; stabbing food to help humans eat is what forks are for. ‘The barriers to development of cancer are embodied in a teleology: cancer cells have defects in regulatory circuits that govern normal cell proliferation and homeostasis.’

Question 32

Hallmarks of Cancer. Acquired Capability: Apoptosis evasion. Conceptual classification of the components of the apoptotic machinery into sensors and effectors. Role of sensors and effectors? Examples of cell surface sensors and survival and death ligand/receptor pairs.

Answer 32

The apoptotic machinery can be broadly divided into two classes of components—sensors and effectors. 
Sensors: monitor the extracellular and intracellular environment for conditions of normality or abnormality that influence whether a cell should live or die. These signals regulate the second class of components, which function as effectors of apoptotic death. 

Examples of cell surface receptors that bind survival or death factors:

1. Survival signal ligand/receptor pairs:
   i) IGF-1/IGF-2 through their receptor, IGF-1R
   ii) IL-3 and its cognate receptor, IL-3R
2. Death signal ligand/receptor pairs:
   i) FAS ligand binding the FAS receptor;
   ii) TNF-alpha binding TNF-R1.

Further, the life of most cells is in part maintained by cell–matrix and cell–cell adherence-based survival signals are needed to maintain life of most cells, and their abrogation elicits apoptosis.

Both soluble and immobilized apoptotic regulatory signals likely reflect the needs of tissues to maintain their constituent cells in appropriate architectural configurations.

Question 33

Hallmarks of Cancer. Acquired Capability: Apoptosis evasion. Conceptual classification of the components of the apoptotic machinery into sensors and effectors. What sort of stimuli have the capacity to trigger intracellular sensors that activate apoptosis?

Answer 33

Intracellular sensors monitor the cell’s well-being and activate the death pathway in response to detecting abnormalities, including

a) DNA damage;
b) signaling imbalance provoked by oncogene action, survival factor insufficiency, or hypoxia.

Question 34

Hallmarks of Cancer. Acquired Capability: Apoptosis evasion. On which organelle do most apoptosis-eliciting signals converge?

Answer 34

Many of the signals that elicit apoptosis converge on the mitochondria, which respond to proapoptotic signals by releasing cytochrome C, a potent catalyst of apoptosis.

Question 35

Hallmarks of Cancer. Acquired Capability: Apoptosis evasion. Bcl-2 family of proteins: Give examples of proapoptotic and antiapoptotic members.

Answer 35

Proapoptotic: Bax, Bak, Bid, Bim
Antiapoptotic: Bcl-2, Bcl-XL, Bcl-W

Question 36

Hallmarks of Cancer. Acquired Capability: Apoptosis evasion. How do Bcl-2 family members and p53 directly instigate apoptosis?

Answer 36

Members of the Bcl-2 family of proteins act in part by governing mitochondrial death signaling through cytochrome C release. The p53 tumor suppressor protein can elicit apo-ptosis by upregulating expression of proapoptotic Bax in response to sensing DNA damage; Bax in turn stimulates mitochondria to release cytochrome C.

Question 37

Hallmarks of Cancer. Acquired Capability: Apoptosis evasion. Which molecules are the ultimate effectors of apoptosis? Which are the ‘gatekeeper’ members of this family and how are they activated?

Answer 37

The ultimate effectors of apoptosis include an array of intracellular proteases termed caspases. Two “gatekeeper” caspases, -8 and -9, are activated by death receptors such as FAS or by the cytochrome C released from mitochondria, respectively.

These proximal caspases trigger the activation of a dozen or more effector caspases that execute the death program, through selective destruction of subcellular structures and organelles, and of the genome.

Question 38

Hallmarks of Cancer. Acquired Capability: Apoptosis evasion. Summarise 4 lines of evidence leading to hypothesis that apoptosis serves as a barrier to cancer

Answer 38

1. The possibility that apoptosis serves as a barrier to cancer was first raised in 1972, when Kerr, Wyllie, and Currie described massive apoptosis in the cells populating rapidly growing, hormone-dependent tumors following hormone withdrawal.
2. Study of bcl-2 and its interaction with myc in inducing lymphoma. The discovery of the bcl-2 oncogene by its upregulation via chromosomal translocation in follicular lymphoma and its recognition as having antiapoptotic activity opened up the investigation of apoptosis in cancer at the molecular level. When coexpressed with a myc oncogene in transgenic mice, the bcl-2 gene was able to promote formation of B cell lymphomas by enhancing lymphocyte survival, not by further stimulating their myc-induced proliferation; further, 50% of the infrequent lymphomas arising in bcl-2 single transgenic transgenic mice had somatic translocations activating c-myc, confirming a selective pressure during lymphomagenesis to upregulate both Bcl-2 and c-Myc. Further insight into the myc-bcl-2 interaction emerged later from studying the effects of a myc oncogene on fibroblasts cultured in low serum. Widespread apoptosis was induced in myc-expressing cells lacking serum; the consequent apoptosis could be abrogated by exogenous survival factors (e.g., IGF-1), by forced overexpression of Bcl-2 or the related Bcl-XL protein, or by disruption of the FAS death signaling circuit. Collectively, the data indicate that a cell’s apo-ptotic program can be triggered by an overexpressed oncogene. Indeed, elimination of cells bearing activated oncogenes by apoptosis may represent the primary means by which such mutant cells are continually culled from the body’s tissues.
3. Study of pRB and p53 in retinoblastoma. In transgenic mice where the pRb tumor suppressor was functionally inactivated in the choroid plexus, slowly growing microscopic tumors arose, exhibiting high apoptotic rates; the additional inactivation of the p53 tumor suppressor protein, a component of the apoptotic signaling circuitry, led to rapidly growing tumors containing low numbers of apoptotic cells.
4. IGF-2 and islet-cell tumours. The role of extracellular survival factors is illustrated by disease progression in transgenic mice prone to pancreatic islet tumors. If IGF-2 gene expression, which is activated in this tumorigenesis pathway, was abrogated using gene knockout mice, tumor growth and progression were impaired, as evidenced by the appearance of comparatively small, benign tumors showing high rates of apoptosis. In these cells, the absence of IGF-2 did not affect cell proliferation rates, clearly identifying it as an antiapoptotic survival factor.

Question 39

Hallmarks of Cancer. Acquired Capability: Apoptosis evasion. Resistance to apoptosis can be achieved by loss of proapoptotic regulators or exploitation of pro-apoptotic pathways. Most commonly lost proapoptotic regulator in cancer? Most commonly subverted anti-apoptotic pathway? Give three known mechanisms for the latter? Describe one mechanism by which cancers can abrogate FAS death signaling?

Answer 39

1. The most commonly occurring loss of a proapoptotic regulator through mutation involves the p53 tumor suppressor gene. The resulting functional inactivation of its product, the p53 protein, is seen in greater than 50% of human cancers. Results in removal of a key component of the DNA damage sensor that can induce the apoptotic effector cascade. Signals evoked by abnormalities, including hypoxia and oncogene hyper-expression, are also funneled in part via p53 to the apo-ptotic machinery; these too are impaired at eliciting apoptosis when p53 function is lost.
2. The PI3 kinase–AKT/PKB pathway, which transmits antiapoptotic survival signals, is likely involved in mitigating apoptosis in a substantial fraction of human tumors. This survival signaling circuit can be activated by
   a) extracellular factors such as IGF-1/2 or IL-3;
   b) intracellular signals emanating from Ras
   c) loss of the pTEN tumor suppressor, a phospholipid phosphatase that normally attenuates the AKT survival signal

FAS death signaling evasion: some cancers can upregulate a nonsignaling decoy receptor for FAS ligand, titrating the death-inducing signal away from the FAS death receptor

Question 40

Hallmarks of Cancer. Acquired Capability: Apoptosis evasion. Discuss the

Answer 40

Most regulatory and effector components are present in redundant form. This redundancy holds important implications for the development of novel types of antitumor therapy, since tumor cells that have lost proapoptotic components are likely to retain other similar ones. We anticipate that new technologies will be able to display the apoptotic pathways still operative in specific types of cancer cells and that new drugs will enable cross-talk between the still intact components of parallel apoptotic signaling pathways in tumor cells, resulting in restoration of the apoptotic defense mechanism, with substantial therapeutic benefit.