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1

The literature on the molecular basis of cancer continues to proliferate at such a rapid pace
that it is easy to get lost in the growing forest of information.

We list some fundamental
principles before delving into the details of the molecular basis of cancer.

  • Nonlethal genetic damage lies at the heart of carcinogenesis
  • A tumor is formed by the clonal expansion of a single precursor cell that has incurred
    genetic damage
    (i.e., tumors are monoclonal).
  • Four classes of normal regulatory genesthe growth-promoting proto-oncogenes, the
    growth-inhibiting tumor suppressor genes, genes that regulate programmed cell death
    (apoptosis), and genes involved in DNA repai
    r—are the principal targets of genetic
    damage.
  • Carcinogenesis is a multistep process at both the phenotypic and the genetic levels,
    resulting from the accumulation of multiple mutations

2

What his the nonlethal genetic damage of carcinogenesis?

Nonlethal genetic damage lies at the heart of carcinogenesis .

Such genetic damage (or
mutation) may be acquired by the action of environmental agents, such as chemicals,
radiation, or viruses, or it may be inherited in the germ line.
[26]

The term
environmental, used in this context, involves any acquired defect caused by exogenous
agents or endogenous products of cell metabolism.
Not all mutations, however, are
“environmentally” induced. Some may be spontaneous and stochastic, falling into the
category of bad luck.

3

The
most commonly used method to determine tumor clonality involves the :

analysis of
methylation patterns adjacent to the highly polymorphic locus of the human androgen
receptor gene, AR. [27]

The frequency of such polymorphisms in the general population
is more than 90%, so it is easy to establish clonality by showing that all the cells in a
tumor express the same allele. For tumors with acquired cytogenetic aberrations of any
type (e.g., a translocation) their presence can be taken as evidence that the
proliferation is clonal.

 

Immunoglobulin receptor and T-cell receptor gene
rearrangements serve as markers of clonality in B- and T-cell lymphomas, respectively.

4

A tumor is formed by the clonal expansion of a single precursor cell that has incurred
genetic damage (i.e., tumors are monoclonal)

 

T or F

True

 

Clonality of tumors can be assessed in
women who are heterozygous for polymorphic X-linked markers, such as the androgen
receptor.

 

 

5

Four classes of normal regulatory genes—

  • the growth-promoting proto-oncogenes,
  • the growth-inhibiting tumor suppressor genes,
  • genes that regulate programmed cell death
  • (apoptosis),
  • and genes involved in DNA repair

are the principal targets of genetic damage.

6

Mutant alleles of proto-oncogenes are considered

 what type of phenotyple?

dominant

 , because they transform cells despite the presence of a normal counterpart.

7

In contrast, typically, both
normal alleles of the tumor suppressor genes
must be damaged before transformation
can occur.

 

T or F

 

 

T

However, there are exceptions to this rule; sometimes, loss of a single allele
of a tumor suppressor gene reduces levels or activity of the protein enough that the
brakes on cell proliferation and survival are released

8

WHat is haploinsufficiency.

Loss of gene function caused by
damage to a single allele is called haploinsufficiency.

 

Such a finding indicates that
dosage of the gene is important
, and that two copies are required for normal
function.

9

Genes that regulate apoptosis may behave as :

proto-oncogenes or tumor
suppressor genes.

10

Mutations of DNA repair genes  directly transform cells by
affecting proliferation or apoptosis.

 

T or F

 

FALSE

 Instead, DNA-repair genes affect cell proliferation or
survival indirectly by influencing the ability of the organism to repair nonlethal damage in
other genes, including proto-oncogenes, tumor suppressor genes, and genes that
regulate apoptosis.

A disability in the DNA-repair genes can predispose cells to
widespread mutations in the genome and thus to
neoplastic transformation.

11

What is a mutator phenotype?

Cells with mutations in DNA repair genes are said to have developed a mutator phenotype. [29]
 

12

Wha is microRNAs (miRNAs)?

Interestingly, a new class of regulatory molecules, called microRNAs (miRNAs), has
recently been discovered ( Chapter 5 ).

Even though they do not encode proteins,
different families of miRNAs have been shown to act as either oncogenes or tumor
suppressors. [29,] [30]

They do so by affecting the translation of other genes as will be
discussed later.

13

Carcinogenesis is a multistep process at both the phenotypic and the genetic levels,
resulting from the accumulation of multiple mutations.

 

T or F

T

14

explain the phenomenon of tumor progression?

malignant neoplasms have several phenotypic attributes, such as excessive growth,
local invasiveness, and the ability to form distant metastases
.

 

Furthermore, it is well
established that over a period of time many tumors become more aggressive and
acquire greater malignant potential.

 

This phenomenon is referred to as tumor
progression and is not simply a function of an increase in tumor size

15

Careful clinical and
experimental studies reveal that increasing malignancy is often acquired in an
incremental fashio
n.

At the molecular level, tumor progression and associated
heterogeneity most likely result from multiple mutations
that accumulate independently
in different cells
, generating subclones with varying abilities to grow, invade,
metastasize, and resist (or respond to) therapy

16

Some of the mutations may
be lethal; others may spur cell growth by affecting additional proto-oncogenes or tumor
suppressor genes.

17

Even though most malignant tumors are monoclonal in origin, by the time they become clinically evident their constituent cells are extremely heterogeneous

18

During progression, tumor cells are subjected to immune and nonimmune selection
pressures.

For example, cells that are highly antigenic are destroyed by host defenses,
whereas those with reduced growth factor requirements are positively selected.

A
growing tumor therefore tends to be enriched for subclones that “beat the odds” and
are adept at survival, growth, invasion, and metastasis

19

FIGURE 7-23 The use of X-linked markers as evidence of the monoclonality of neoplasms.
Because of random X inactivation, all females are mosaics with two cell populations (with
different alleles for the androgen receptor labeled A and B in this case).

When neoplasms
that arise in women who are heterozygous for X-linked markers are analyzed, they are made
up of cells that contain the active maternal (XA) or the paternal (XB) X chromosome but not
both.

20

Q image thumb

FIGURE 7-24 Tumor progression and generation of heterogeneity. New subclones arise
from the descendants of the original transformed cell by multiple mutations. With progression
the tumor mass becomes enriched for variants that are more adept at evading host defenses
and are likely to be more aggressive

21

It is traditional to describe cancerassociated
genes on the basis of their presumed function. It is beneficial, however, to consider
cancer-related genes in the context of seven fundamental changes in cell physiology that
together determine malignant phenotype.
[32] (Another important change for tumor
development is escape from immune attack . This property is discussed later in this chapter.)
The seven key changes are the following:

  • Self-sufficiency in growth signals
  • Insensitivity to growth-inhibitory signals
  • Evasion of apoptosis
  • Limitless replicative potential
  • Sustained angiogenesis:
  • Ability to invade and metastasize
  • Defects in DNA repair

22

Explain Self-sufficiency in growth signals:

Self-sufficiency in growth signals:

Tumors have the capacity to proliferate without
external stimuli
, usually as a consequence of oncogene activation.
 

23

 Explain Insensitivity to growth-inhibitory signals : 

Tumors may not respond to molecules that are
inhibitory to the proliferation of normal cells such as transforming growth factor β (TGF-
β) and direct inhibitors of cyclin-dependent kinases (CDKIs).

24

Explain Evasion of apoptosis

Evasion of apoptosis:

Tumors may be resistant to programmed cell death, as a
consequence of inactivation of p53 or activation of anti-apoptotic genes.

25

Explain the Limitless replicative potential: 

Tumor cells have unrestricted proliferative capacity,
avoiding cellular senescence and mitotic catastrophe.

26

Tumor cells, like normal cells, are not able to grow without
formation of a vascular supply to bring nutrients and oxygen and remove waste
products.

T or F

 

True

 

Sustained angiogenesis

 Hence, tumors must induce angiogenesis.

27

 

Tumors may fail to repair DNA damage caused by carcinogens
or incurred during unregulated cellular proliferation, leading to genomic instability and
mutations in proto-oncogenes and tumor suppressor genes.

 

T or F

 

True

Defects in DNA repair 

28

Mutations in one or more genes that regulate these cellular traits are seen in every cancer.
However, the precise genetic pathways that give rise to these attributes differ between
individual cancers, even within the same organ.

29

Mutations in one or more genes that regulate these cellular traits are seen in every cancer.
However, the precise genetic pathways that give rise to these attributes differ between
individual cancers, even within the same organ.

 

T or F

T

30

Indeed, recent studies in a variety of human
tumors, such as melanoma and prostate adenocarcinoma, have shown that oncogene-induced
senescence,
wherein mutation of a proto-oncogene drives cells into senescence rather than
proliferation,
is an important barrier to carcinogenesis. [33]

Some limits to neoplastic growth are
even physical.

If a tumor is to grow larger than 1 to 2 mm, mechanisms that allow the delivery of
nutrients and the elimination of waste products must be provided (angiogenesis).

Furthermore,
epithelia are separated from the interstitial matrix by a basement membrane, composed of
extracellular matrix molecules, that must be broken down by invasive carcinoma cells.

These
protective barriers, both intrinsic and extrinsic to the cell, must be breached, and feedback
loops that normally prevent uncontrolled cell division must be disabled by mutations before a
fully malignant tumor can emerge

31

Q image thumb

FIGURE 7-25 Flowchart depicting a simplified scheme of the molecular basis of cancer.
 

32

Define oncogenes.

Genes that promote autonomous cell growth in cancer cells are called oncogenes

33

Define proto-oncogene,

Genes that promote autonomous cell growth in cancer cells are called oncogenes, and their
unmutated cellular counterparts are called proto-oncogenes

34

Oncogenes are created by
mutations in proto-oncogenes
and are characterized by the ability to promote cell growth in the
absence of normal growth-promoting signals

35

What are oncoproteins?

Oncogenes are created by
mutations in proto-oncogenes and are characterized by the ability to promote cell growth in the
absence of normal growth-promoting signals.

Their products, called oncoproteins, resemble the
normal products of proto-oncogenes except that oncoproteins are often devoid of important

internal regulatory elements, and their production in the transformed cells does not depend on
growth factors or other external signals.

In this way cell growth becomes autonomous, freed
from checkpoints and dependence upon external signals.

36

To aid in the understanding of the
nature and functions of oncoproteins and their role in cancer, it is necessary to briefly mention
the sequential steps that characterize normal cell proliferation. Under physiologic conditions cell
proliferation can be readily resolved into the following steps:

  • The binding of a growth factor to its specific receptor
  • Transient and limited activation of the growth factor receptor, which, in turn, activates several signal-transducing proteins on the inner leaflet of the plasma membrane
  •  Transmission of the transduced signal across the cytosol to the nucleus via second messengers or by a cascade of signal transduction molecules
  •  Induction and activation of nuclear regulatory factors that initiate DNA transcription
  •  Entry and progression of the cell into the cell cycle, ultimately resulting in cell division

37

Proto-oncogenes have multiple roles, participating in cellular functions related to growth and
proliferation.

Proteins encoded by proto-oncogenes may function as :

  • growth factors
  • or their receptors,
  • signal transducers,
  • transcription factors,
  • or cell cycle components.

38

Oncoproteins
encoded by oncogenes generally serve functions similar to their normal counterparts ( Table 7-
6 ). However, mutations convert proto-oncogenes into constitutively active cellular oncogenes
that are involved in tumor development because the oncoproteins they encode endow the cell
with self-sufficiency in growth

39

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

 

GROWTH FACTORS

PDGF-β chain

 

Protooncogene: Mode of Activation :Associated Human Tumor

 

SIS (official name PBGFB):Overexpression:Astrocytoma Osteosarcoma

40

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

 

GROWTH FACTORS

Fibroblast growth
factors

Protooncogene: Mode of Activation :Associated Human Tumor

 

HST1:Overexpression:Stomach cancer

INT2 (official name FGF3):Amplification:Bladder cancer,Breast cancerMelanoma

41

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

 

GROWTH FACTORS

TGF-α

 

Protooncogene: Mode of Activation :Associated Human Tumor

 

TGFA:Overexpression:Astrocytomas, Hepatocellular carcinomas

 

42

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

 

GROWTH FACTORS

HGF

Protooncogene: Mode of Activation :Associated Human Tumor

 

HGF: Overexpression: Thyroid cancer

43

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

 

GROWTH FACTOR RECEPTORS

EGF-receptor
famil

Protooncogene: Mode of Activation :Associated Human Tumor

 

ERBB1(EGFR), ERRB2:  Overexpression: Squamous cell carcinoma of lung, gliomas

44

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

 

GROWTH FACTOR RECEPTORS

FMS-like tyrosine
kinase 3

 

Protooncogene: Mode of Activation :Associated Human Tumor

FLT3: Amplification: Breast and ovarian cancers

45

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

Receptor for
neurotrophic
factors

Protooncogene: Mode of Activation :Associated Human Tumor

 

RET :Point mutation: Leukemia

46

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

GROWTH FACTOR RECEPTORS

 

Receptor for
neurotrophic
factors

Protooncogene: Mode of Activation :Associated Human Tumor

 

RET:Point mutation: Leukemia

47

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

GROWTH FACTOR RECEPTORS

 

Receptor for
neurotrophic
factors

 

Protooncogene: Mode of Activation :Associated Human Tumor

 

RET:Point mutation :Multiple endocrine neoplasia 2A and B, familial medullary thyroid carcinomas

48

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

GROWTH FACTOR RECEPTORS

 

PDGF receptor

Protooncogene: Mode of Activation :Associated Human Tumor

 

PDGFRB Overexpression,translocation: Gliomas, lekemias

49

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

GROWTH FACTOR RECEPTORS

 

Receptor for stem
cell (steel) factor

Protooncogene: Mode of Activation :Associated Human Tumor

 

KIT: Point mutation: Gastrointestinal stromal tumors,
seminomas, leukemias

50

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

PROTEINS INVOLVED IN SIGNAL TRANSDUCTION

 

GTP-binding

 

Protooncogene: Mode of Activation :Associated Human Tumor

KRAS: Point mutation: Colon, lung, and pancreatic tumors

51

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

PROTEINS INVOLVED IN SIGNAL TRANSDUCTION

 

GTP-binding

 

 

 

Protooncogene: Mode of Activation :Associated Human Tumor

HRAS :Point mutation: Bladder and kidney tumors

52

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

PROTEINS INVOLVED IN SIGNAL TRANSDUCTION

 

GTP-binding

 

 

Protooncogene: Mode of Activation :Associated Human Tumor

NRAS: Point mutation :Melanomas, hematologic malignancies

53

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

PROTEINS INVOLVED IN SIGNAL TRANSDUCTION

 

Nonreceptor
tyrosine kinase

 

 

Protooncogene: Mode of Activation :Associated Human Tumor

 

ABL :Translocation: Chronic myeloid leukemia
Acute lymphoblastic leukemia

54

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

PROTEINS INVOLVED IN SIGNAL TRANSDUCTION

 

RAS signal
transduction

 

 

Protooncogene: Mode of Activation :Associated Human Tumor

BRAF :Point mutation: Melanomas

55

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

PROTEINS INVOLVED IN SIGNAL TRANSDUCTION

 

WNT signal
transduction

 

 

Protooncogene: Mode of Activation :Associated Human Tumor

 

β-catenin: Point mutation,Overexpression :Hepatoblastomas, hepatocellular carcinoma

56

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

 

NUCLEAR-REGULATORY PROTEINS

Transcriptional
activators

 

Protooncogene: Mode of Activation :Associated Human Tumor

C-MYC: Translocation: Burkitt lymphoma

57

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

 

NUCLEAR-REGULATORY PROTEINS

Transcriptional
activators

 

 

Protooncogene: Mode of Activation :Associated Human Tumor

 

N-MYC: Amplification: Neuroblastoma, small-cell carcinoma of lung

58

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

 

NUCLEAR-REGULATORY PROTEINS

Transcriptional
activators

 

Protooncogene: Mode of Activation :Associated Human Tumor

 

 

L-MYC: Amplification: Small-cell carcinoma of lung

59

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

 

CELL CYCLE REGULATORS

Cyclins

 

Protooncogene: Mode of Activation :Associated Human Tumora

 

Cyclin D :Translocation :Mantle cell lymphoma

60

 

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

 

CELL CYCLE REGULATORS

Cyclins

 

Protooncogene: Mode of Activation :Associated Human Tumor

 

Cyclin D: Amplification: Breast and esophageal cancers

61

 

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

 

CELL CYCLE REGULATORS

Cyclins

 

 

Protooncogene: Mode of Activation :Associated Human Tumor

 

Cyclin E :Overexpression: Breast cancer

62

 

TABLE 7-6 -- Selected Oncogenes, Their Mode of Activation, and Associated Human
Tumors

 

 

CELL CYCLE REGULATORS

Cyclin-dependent
kinase

 

Protooncogene: Mode of Activation :Associated Human Tumor

 

CDK4 :Amplification or point mutation:
Glioblastoma, melanoma, sarcoma

63

Growth Factors.

 

Normal cells require stimulation by growth factors to undergo proliferation.

 

Most soluble growth
factors are made by one cell type and act on a neighboring cell to stimulate proliferation
(paracrine action).

 

Many cancer cells, however, acquire the ability to synthesize the same
growth factors to which they are responsive, generating an autocrine loop
.

 

For example, many
glioblastomas secrete platelet-derived growth factor (PDGF) and express the PDGF receptor,
and many sarcomas make both transforming growth factor α (TGF-α) and its receptor.

Although
an autocrine loop is considered to be an important element in the pathogenesis of several
tumors, in most instances the growth factor gene itself is not altered or mutated.

More
commonly, products of other oncogenes that lie along many signal transduction pathways, such
as RAS, cause overexpression of growth factor genes, thus forcing the cells to secrete large
amounts of growth factors, such as TGF-α. Nevertheless, increased growth factor production is
not sufficient for neoplastic transformation

In all likelihood growth factor driven proliferation
contributes to the malignant phenotype
by increasing the risk of spontaneous or induced
mutations in the proliferating cell population.

64

Growth Factor Receptors.

 

Several oncogenes that encode growth factor receptors have been found.

To understand how
mutations affect the function of these receptors, it should be recalled that one important class
of growth factor receptors are transmembrane proteins with an external ligand-binding domain
and a cytoplasmic tyrosine kinase domain
( Chapter 3 ).

 

In the normal forms of these receptors,
the kinase is transiently activated by binding of the specific growth factors, followed rapidly by
receptor dimerization and tyrosine phosphorylation of several substrates that are a part of the
signaling cascade.

What is the oncogenic verison of these?

The oncogenic versions of these receptors are associated with constitutive
dimerization and activation without binding to the growth factor

. Hence, the mutant receptors
deliver continuous mitogenic signals to the cell, even in the absence of growth factor in the
environment.

65

Growth factor receptors can be constitutively activated in tumors by multiple different
mechanisms, including:

 mutations, gene rearrangements, and overexpression

66

What is RET protooncogene?
 

a receptor tyrosine kinase, exemplifies oncogenic conversion via mutations and
gene rearrangements. [33]

 

 

67

RET protein is a receptor for:

The RET protein is a receptor for the glial cell line–derived
neurotrophic factor and structurally related proteins that promote cell survival during neural
development.

 

RET is normally expressed in neuroendocrine cells, such as parafollicular C cells
of the thyroid, adrenal medulla, and parathyroid cell precursors.

68

Point mutations in the RET
proto-oncogene are associated with :

dominantly inherited MEN types 2A and 2B and familial
medullary thyroid carcinoma ( Chapter 24 ).

 

 

69

What is In MEN-2A?

 

point mutations in the RET extracellular
domain
cause constitutive dimerization and activation, leading to medullary thyroid carcinomas
and adrenal and parathyroid tumors. 

 

MEN 2A loves to TAP

 

70

\What is MEN- 2B mutation?

In MEN-2B, point mutations in the RET cytoplasmic
catalytic domain alter the substrate specificity of the tyrosine kinase and lead to thyroid and
adrenal tumors without involvement of the parathyroid.

 

"MEN 2B- BONELESS"

 

In all these familial conditions, the
affected individuals inherit the RET mutation in the germline.

Sporadic medullary carcinomas of
the thyroid are associated with somatic rearrangements of the RET gene, generally similar to
those found in MEN-2B.

71

What is point mutation in FLT3?

Point mutations in FLT3, the gene encoding the FMS-like tyrosine kinase
3 receptor, that lead to constitutive signaling have been detected in myeloid leukemias.

72

In
certain chronic myelomonocytic leukemias with the (5;12) translocation, the entire cytoplasmic
domain of the PDGF receptor is fused with a segment of an ETS family trancription factor,
resulting in permanent dimerization of the PDGF receptor.

 

 

73

Greater than 90% of gastrointestinal
stromal tumors have a constitutively activating mutation in the r___________, 

eceptor tyrosine kinase c-KIT or
PDGFR

 

which are the receptors for stem cell factor and PDGF, respectively.

These mutations
are amenable to specific inhibition by the tyrosine kinase inhibitor imatinib mesylate.
This type
of therapy, directed to a specific alteration in the cancer cell, is called targeted therapy .

74

Far more common than mutations of these proto-oncogenes is overexpression of normal forms
of growth factor receptors.

 

In some tumors increased receptor expression results from gene
amplification
, but in many cases the molecular basis of increased receptor expression is not
fully know

Two members of the _____________- are the best
described

epidermal growth factor (EGF) receptor family

75

What is ERBB1 

The normal form of ERBB1, the EGF receptor gene, is overexpressed in up to 80%
of squamous cell carcinomas of the lung
, in 50% or more of glioblastomas ( Chapter 28 ), and
in 80% to 100% of head and neck tumors. [38,] [39]

76

What is ERBB2?

Likewise, the ERBB2 gene (also called
HER-2/NEU
), the second member of the EGF receptor family, is amplified in approximately 25%
of breast cancers and in human adenocarcinomas arising within the ovary, lung, stomach, and
salivary glands.
[36]

 

GLANDS!!!

 

Because the molecular alteration in ERBB2 is specific for the cancer cells,
new therapeutic agents consisting of monoclonal antibodies specific to ERBB2 have been
developed and are currently in use clinically, providing yet another example of targeted

therapy.

77

Signal-Transducing Proteins.

 

Several examples of oncoproteins that mimic the function of normal cytoplasmic signal- transducing proteins have been found.

Most such proteins are strategically located on the inner leaflet of the plasma membrane, where they receive signals from outside the cell (e.g., by
activation of growth factor receptors) and transmit them to the cell's nucleus.

Biochemically, the
signal-transducing proteins are heterogeneous.

The most well-studied example of a signaltransducing
oncoprotein is the :

RAS family of guanine triphosphate (GTP)-binding proteins (G
proteins).

78

The RAS genes, of which there are three in the human genome (________________), were
discovered initially in transforming retroviruses

HRAS, KRAS, NRAS)

79

What is the single
most common abnormality of proto-oncogenes in human tumors?

Point mutation of RAS family genes is the single
most common abnormality of proto-oncogenes in human tumors

80

Approximately 15% to 20% of
all human tumors contain mutated versions of RAS proteins. [40]

 

The frequency of such
mutations varies with different tumors, but in some types of cancers it is very high. For example,
 

90% of pancreatic adenocarcinomas and cholangiocarcinomas contain a RAS point mutation,
as do about 50% of colon, endometrial, and thyroid cancers and 30% of lung adenocarcinomas
and myeloid leukemias

81

In general, carcinomas of the three types of RAS :

In general, carcinomas :

 

  • (particularly from colon and pancreas) have mutations of KRAS
  •  bladder tumors have HRAS mutations,
  •  hematopoietic tumors bear NRAS mutations.

 

RAS mutations are infrequent in certain other cancers, such as those arising

in the uterine cervix or breast

82

What is the role of RAS?

RAS plays an important role in signaling cascades downstream of growth factor receptors,
resulting in mitogenesis.

 

For example, abrogation of RAS function blocks the proliferative
response to EGF, PDGF, and CSF-1.

83

How is the normal RAS?

Normal RAS proteins are tethered to the cytoplasmic
aspect of the plasma membrane, as well as the endoplasmic reticulum and Golgi membranes.
They can be activated by growth factor binding to receptors at the plasma membrane. [40]

RAS
is a member of a family of small G proteins that bind guanosine nucleotides (guanosine
triphosphate, GTP and guanosine diphosphate, GDP), similar to the larger trimolecular G
proteins.

Normally RAS proteins flip back and forth between an excited signal-transmitting state
and a quiescent state.

In the inactive state, RAS proteins bind GDP. Stimulation of cells by
growth factors leads to exchange of GDP for GTP and subsequent conformational changes that
generates active RAS ( Fig. 7-26 ).

The activated RAS stimulates downstream regulators of proliferation, such as the mitogen-activated protein (MAP) kinase cascade , which floods the
nucleus with signals for cell proliferation.

84

FIGURE 7-26 Model for action of RAS genes.

 

 

When a normal cell is stimulated through a
growth factor receptor, inactive (GDP-bound) RAS is activated to a GTP-bound state.
Activated RAS recruits RAF and stimulates the MAP-kinase pathway to transmit growthpromoting
signals to the nucleus. The mutated RAS protein is permanently activated
because of inability to hydrolyze GTP, leading to continuous stimulation of cells without any
external trigger. The anchoring of RAS to the cell membrane by the farnesyl moiety is
essential for its action. See text for explanation of abbreviations.

85

The orderly cycling of the RAS protein depends on two reactions: 

(1) nucleotide exchange (GDP by GTP), which activates RAS protein, and

 

(2) GTP hydrolysis, which converts the GTPbound,
active RAS to the GDP-bound, inactive form. Both these processes are extrinsically
regulated by other proteins.

86

The removal of GDP and its replacement by GTP during RAS activation are catalyzed by a family of guanine nucleotide–releasing proteins.

 

Conversely, the
GTPase activity intrinsic to normal RAS proteins is dramatically accelerated by GTPaseactivating
proteins (GAPs).
These widely distributed proteins bind to the active RAS and
augment its GTPase activity by more than 1000-fold, leading to termination of signal
transduction. Thus, GAPs function as “brakes” that prevent uncontrolled RAS activity.

87

Several distinct point mutations of RAS have been identified in cancer cells.

The affected
residues lie within either :

the GTP-binding pocket or the enzymatic region essential for GTP
hydrolysis, and thus markedly reduce the GTPase activity of the RAS protein. 

 

Mutated RAS is
trapped in its activated GTP-bound form, and the cell is forced into a continuously proliferating
state. It follows from this scenario that the consequences of mutations in RAS protein would be
mimicked by mutations in the GAPs that fail to activate the GTPase activity and thus restrain
normal RAS proteins. Indeed, disabling mutation of neurofibromin 1, a GAP, is associated with
the inherited cancer syndrome familial neurofibromatosis type 1 ( Chapter 27 ).

88

In addition to RAS, downstream members of the RAS signaling cascade (RAS/RAF/MAP kinase)
may also be altered in cancer cells, resulting in a similar phenotype.

 

Thus, mutations in ____________-,
one of the members of the RAF family, have been detected in more than 60% of melanomas
and in more than 80% of benign nevi.
[44,] [45]

 

BRAF

 

This suggests that dysregulation of the
RAS/RAF/MAP kinase pathway may be one of the initiating events in the development of
melanomas, although it is not sufficient by itself to cause tumorigenesis. Indeed, BRAF
mutations alone lead to oncogene-induced senescence giving rise to benign nevi rather than
malignant melanoma. Thus, oncogene-induced senescence is a barrier to carcinogenesis that
must be overcome by mutation and disabling of key protective mechanisms, such as those
provided by the p53 gene (discussed later).

89

Alterations in Nonreceptor Tyrosine Kinases

 

Mutations that unleash latent oncogenic activity occur in several non-receptor-associated
tyrosine kinases,
which normally function in signal transduction pathways that regulate cell
growth
( Chapter 3 ).

As with receptor tyrosine kinases, in some instances the mutations take
the form of chromosomal translocations or rearrangements that create fusion genes encoding
constitutively active tyrosine kinases.

An important example of this oncogenic mechanism
involves the______________

 c-ABL tyrosine kinase

90

In ____________ the ABL
gene is translocated from its normal abode on chromosome 9 to chromosome 22
( Fig. 7-27 ),
where it fuses with the BCR gene (see discussion of chromosomal translocations, later in this
chapter). The resultant chimeric gene encodes a constitutively active, oncogenic BCR-ABL
tyrosine kinase.

CML and some acute lymphoblastic leukemias,

91

Several structural features of the BCR-ABL fusion protein contribute to the
increased kinase activity, but the most important is that ____________

 

the BCR moiety promotes the selfassociation
of BCR-ABL. 

 

This is a common theme, since many different oncogenic tyrosine
kinases consist of fusion proteins in which the non–tyrosine kinase partner drives selfassociation.

92

Treatment of CML has been revolutionized by the development of____________

 imatinib
mesylate, a “designer” drug with low toxicity and high therapeutic efficacy that inhibits the BCRABL
kinase. [47] [48] [49]

 

This is another example of rational drug design emerging from an
understanding of the molecular basis of cancer. It is also an example of the concept of
oncogene addiction.

 

Despite accumulation of numerous mutations throughout the genome,
signaling through the BCR-ABL gene is required for the tumor to persist, hence inhibition of its
activity is effective therapy.

93

___________ is an early, perhaps initiating event, during
leukemogenesis.

 

BCR-ABL translocation

 

The remaining mutations are selected for, and built around, the constant
signaling through BCR-ABL.

BCR-ABL signaling can be seen as the central lodgepole around
which the structure is built.

Remove the lodgepole by inhibition of the BCR-ABL kinase, and the
structure collapse

94

In other instances, nonreceptor tyrosine kinases are activated by point mutations that abrogate
the function of negative regulatory domains that normally hold enzyme activity in check.

For
example, several myeloproliferative disorders, particularly polycythemia vera and primary
myelofibrosis
, are highly associated with activating
______________- (
Chapter 13 ). [51] 

point mutations in the tyrosine kinase JAK2

 

The aberrant JAK2 kinase in turn activates transcription factors of the STAT
family, which promote the growth factor–independent proliferation and survival of the tumor
cells.

Recognition of this molecular lesion has led to trials of JAK2 inhibitors in myeloproliferative
disorders, and stimulated searches for activating mutations in other nonreceptor tyrosine
kinases in a wide variety of human cancers.

95

What are Transciption Factors?

 

 

 

 

Just as all roads lead to Rome, all signal transduction pathways converge to the nucleus, where
a large bank of responder genes that orchestrate the cell's orderly advance through the mitotic
cycle are activated.

Indeed, the ultimate consequence of signaling through oncogenes like RAS
or ABL is inappropriate and continuous stimulation of nuclear transcription factors that drive
growth-promoting genes.

 

Transcription factors contain specific amino acid sequences or motifs
that allow them to bind DNA or to dimerize for DNA binding
Binding of these proteins to specific
sequences in the genomic DNA activates transcription of genes.
 

 

96

What happens when theree is a mutations affecting genes that regulate transcription?

Growth autonomy may thus
occur as a consequence of mutations affecting genes that regulate transcription. 

97

A host of
oncoproteins, including products of the _________oncogenes, are
transcription factors that regulate the expression of growth-promoting genes, such as cyclins.
 

MYC, MYB, JUN, FOS, and REL 

 

Of these, MYC is most commonly involved in human tumors , and hence a brief overview of its
function is warranted.

98

What is the MYC Oncogene?

The MYC proto-oncogene is expressed in virtually all eukaryotic cells and belongs to the
immediate early response genes, which are rapidly induced when quiescent cells receive a
signal to divide
(see discussion of liver regeneration in Chapter 3 ).

After a transient increase of
MYC messenger RNA, the expression declines to a basal level.

The molecular basis of MYC
function in cell replication is not entirely clear
.

As with many transcription factors, it is thought that MYC is involved in carcinogenesis by activating genes that are involved in proliferation

99

Indeed, some of MYC target genes, such as ornithine decarboxylase and cyclin D2, are known to
be associated with _______.

 

cell proliferation

100

However, the range of activities modulated by MYC is very broad and includes:

  •  histone acetylation
  • , reduced cell adhesion,
  • increased cell motility
  • , increased telomerase activity,
  • increased protein synthesis,
  • decreased proteinase activity,
  • and other changes in cellular matbolism that enable a high rate of cell division.

101

Genomic mapping of
MYC binding sites has identified thousands of different sites and an equivalent number of
genes that might be regulated. [53]

 

However, there is little overlap in the MYC target genes in different cancers, preventing identification of a canonical MYC carcinogenesis program.
Interestingly, it has been recently suggested that MYC interacts with components of the DNAreplication
machinery, and plays a role in the selection of origins of replication
. [54]

Thus,
overexpression of MYC may drive activation of more __________________

origins than needed for normal cell
division, or bypass checkpoints involved in replication, leading to genomic damage and

accumulation of mutations.

 

Finally, MYC is one of a handful of transcription factors that can act in concert to reprogram somatic cells into pluripotent stem cells ( Chapter 3 );

MYC may also
enhance self-renewal, block differentiation, or both.

102

While on one hand MYC activation is linked to proliferation, on the other hand, cells in culture
undergo apoptosis if MYC activation occurs in the absence of survival signals (growth
factors). [55]

The MYC proto-oncogene contains separate domains that encode the growthpromoting
and apoptotic activities, but it is not clear whether MYC-induced apoptosis occurs in
vivo.

103

In contrast to the regulated expression of MYC during normal cell proliferation, persistent
expression, and in some cases overexpression, of the MYC protein are commonly found in
tumors.

 

Dysregulation of MYC expression resulting from translocation of the gene occurs in
_________ (see Fig. 7-27 ).

 

Burkitt lymphoma, a B-cell tumor

104

MYC is amplified in some cases of breast,
colon, lung, and many other carcinomas.

 

105

The related ____genes are amplified in
neuroblastomas ( Fig. 7-28 ) and small-cell cancers of the lung, respectively.

N-MYC and L-MYC

106

Cyclins and Cyclin-Dependent Kinases

 

The ultimate outcome of all growth-promoting stimuli is the entry of quiescent cells into the cell
cycle.

Cancers may grow autonomously if the genes that drive the cell cycle become
dysregulated by mutations or amplification.

107

What is the rold of CDKs?

the orderly progression
of cells through the various phases of the cell cycle is orchestrated by cyclin-dependent
kinases (CDKs), which are activated by binding to cyclins, so called because of the cyclic
nature of their production and degradation.

 

 

" SIGNAL LIGHT"

108

The CDK-cyclin complexes phosphorylate crucial
target proteins that drive the cell through the cell cycle. On completion of this task, cyclin levels
decline rapidly. More than 15 cyclins have been identified; ______________appear
sequentially during the cell cycle and bind to one or more CDK.

The cell cycle may thus be
seen as a relay race in which each lap is regulated by a distinct set of cyclins, and as one set of
cyclins leaves the track, the next set takes over (

cyclins D, E, A, and B 

 

"BEAD"

 

DNAs are like BEADS

109

Q image thumb

FIGURE 7-29 Schematic illustration of the role of cyclins, CDKs, and CDK inhibitors (CDKIs)
in regulating the cell cycle. The shaded arrows represent the phases of the cell cycle during
which specific cyclin-CDK complexes are active. As illustrated, cyclin D–CDK4, cyclin D
–CDK6, and cyclin E–CDK2 regulate the G1-to-S transition by phosphorylation of the RB
protein (pRB). Cyclin A–CDK2 and cyclin A–CDK1 are active in the S phase. Cyclin B–CDK1
is essential for the G2-to-M transition. Two families of CDKIs can block activity of CDKs and
progression through the cell cycle. The so-called INK4 inhibitors, composed of p16, p15,
p18, and p19, act on cyclin D–CDK4 and cyclin D–CDK6. The other family of three inhibitors,
p21, p27, and p57, can inhibit all CDKs.

110

TABLE 7-7 -- Main Cell Cycle Components and Their Inhibitors

 

CYCLIN-DEPENDENT KINASES

CDK4

CDK2

CDK1

 

111

CDK4

 Forms a complex with cyclin D that phosphorylates RB, allowing the cell to progress
through the G1 restriction point.

112

 CDK2 

Forms a complex with cyclin E in late G1, which is involved in G1/S transition. Forms
a complex with cyclin A at the S phase that facilitates G2/M transition.

113

CDK1 

Forms a complex with cyclin B that facilitates G2/M transition.

114

Cell Cycle
Component

INHIBITORS

  • CIP/KIP family: p21, p27 (CDKN2A-C)
  • INK4/ARF family (CDKN1A-D)

115

CIP/KIP family: p21,
p27 (CDKN2A-C)

Block the cell cycle by binding to cyclin-CDK complexes; p21 is induced by the
tumor suppressor p53; p27 responds to growth suppressors such as TGF-β.

116

INK4/ARF
family (CDKN1A-D)

p16/INK4a binds to cyclin D–CDK4 and promotes the inhibitory effects of RB;
p14/ARF increases p53 levels by inhibiting MDM2 activity.

117

Cell Cycle
Component

 

CHECKPOINT COMPONENTS

p53

 

Ataxiatelangiectasia
mutated

118

p53

Tumor suppressor gene altered in the majority of cancers; causes cell cycle arrest
and apoptosis
.

Acts mainly through p21 to cause cell cycle arrest. Causes apoptosis
by inducing the transcription of pro-apoptotic genes such as BAX .

 p53 is required for the
G1/S checkpoint and is a main component of the G2/M checkpoint.

119

Levels of p53 are
negatively regulated by __________through a feedback loop.

MDM2 

120

Ataxiatelangiectasia
mutated

Activated by mechanisms that sense double-stranded DNA breaks.

Transmits signals to arrest the cell cycle after DNA damage.

 

Acts through p53 in the G1/S
checkpoint. At the G2/M checkpoint, it acts both through p53-dependent mechanisms and through the inactivation of CDC25 phosphatase, which disrupts
the cyclin B–CDK1 complex.

 

Component of a network of genes that include BRCA1
and BRCA2, which link DNA damage with cell cycle arrest and apoptosis.

121

With this background it is easy to appreciate that mutations that dysregulate the activity of
cyclins and CDKs favor cell proliferation
.

 

Mishaps affecting the expression of cyclin D or CDK4
seem to be a common event in neoplastic transformation.

The cyclin D genes are
overexpressed in many cancers, including those _____________. 

affecting the breast, esophagus, liver, and a
subset of lymphomas

122

Amplification of the CDK4 gene occurs in__________ 

 melanomas, sarcomas, and
glioblastomas.

123

Mutations affecting cyclin B and cyclin E and other CDKs also occur, but they are
much less frequent.

124

While cyclins arouse the CDKs, their inhibitors (CDKIs), of which there are many, silence the
CDKs and exert negative control over the cell cycle.

 

T or F

True

125

The CIP/WAF family of CDKIs, composed of
three proteins, called :

 

 inhibits the CDKs
broadly

  • p21 (CDKN1A),
  • p27 (CDKN1B), and
  • p57 (CDKN1C)

126

INK4 family of CDK1s, made up of:__________

 has selective effects on cyclin D/CDK4 and cyclin D/CDK6.

  •  p15 (CDKN2B),
  • p16 (CDKN2A),
  • p18 (CDKN2C), and
  • p19 (CDKN2D)

127

Expression of these CDK inhibitors is down-regulated by mitogenic signaling pathways, thus
promoting the progression of the cell cycle.

 

For example, p27 (CDKN1B), a CDKI that inhibits
cyclin E
, is expressed throughout G1.

Mitogenic signals dampen the activity of p27 in a variety of ways, relieving inhibition of cyclin E-CDK2 and thus allowing the cell cycle to proceed. [56]
The CDKIs are frequently mutated or otherwise silenced in many human malignancies.

128

Germline mutations of________are associated with 25% of melanoma-prone
kindreds
. [23]

 

 

 p16 (CDKN2A) 

129

Somatically acquired deletion or inactivation of p16 is seen in :

  • 75% of pancreatic carcinomas,
  •  40% to 70% of glioblastomas
  • 50% of esophageal cancers,
  • 20% to 70% of acute  lymphoblastic leukemias,and
  • 20% of non-small-cell lung carcinomas, soft-tissue sarcomas, and bladder cancers

130

What are checkpoints?

internal controls of the cell cycle called checkpoints,

131

There are two main cell cycle checkpoints, ____________

  • one at the G1/S transition
  • and the other at G2/M.

132

What is the point of no return in the cell cycle?

The S phase is the point of no return in the cell cycle.

133

What is the role of G1/S checkpoint

Before a cell makes the final commitment
to replicate, the G1/S checkpoint checks for DNA damage; if damage is present, the DNA-repair
machinery and mechanisms that arrest the cell cycle are put in motion.

The delay in cell cycle
progression provides the time needed for DNA repair; if the damage is not repairable, apoptotic
pathways are activated to kill the cell.

 

Thus, the G1/S checkpoint prevents the replication of
cells that have defects in DNA, which would be perpetuated as mutations or chromosomal
breaks in the progeny of the cell.

DNA damaged after its replication can still be repaired as long
as the chromatids have not separated.

134

What is the function of G2/M checkpoint?

The G2/M checkpoint monitors the completion of DNA
replication
and checks whether the cell can safely initiate mitosis and separate sister
chromatids
.

 

This checkpoint is particularly important in cells exposed to ionizing radiation.

Cells
damaged by ionizing radiation activate the G2/M checkpoint and arrest in G2; defects in this
checkpoint give rise to chromosomal abnormalities.

135

To function properly, cell cycle checkpoints
require sensors of DNA damage, signal transducers, and effector molecules. [58]

 

136

The sensors
and transducers of DNA damage seem to be similar for the G1/S and G2/M checkpoints.

 

They
include,

  •  as sensors,
  • proteins of the RAD family
  • and ataxia telangiectasia mutated (ATM) and as transducers,
  • the CHK kinase families. [59]

 

137

The checkpoint effector molecules differ, depending

on the cell cycle stage at which they act.

 

T or F

 

 

T

138

In the G1/S checkpoint, cell cycle arrest is mostly
mediated through________

 

 p53, which induces the cell cycle inhibitor p21. 

 

139

Arrest of the cell cycle by the
G2/M checkpoint involves_______.

Defects in
cell cycle checkpoint components
are a major cause of genetic instability in cancer cells.

 both p53-dependent and p53-independent mechanisms

140

INSENSITIVITY TO GROWTH INHIBITION AND ESCAPE FROM SENESCENCE: 

TUMOR
SUPPRESSOR GENES

141

Failure of growth inhibition is one of the fundamental alterations in the process of
carcinogenesis.

Whereas oncogenes drive the proliferation of cells, the products of tumor
suppressor genes apply _______________

brakes to cell proliferation

142

It has become apparent that
the tumor suppressor proteins form a network of checkpoints that prevent uncontrolled growth.
Many tumor suppressors, such as ________, are part of a regulatory network that recognizes
genotoxic stress from any source, and responds by shutting down proliferation

RB and p53

143

Indeed,
expression of an oncogene in an otherwise completely normal cell leads to quiescence, or to
permanent cell cycle arrest
(oncogene-induced senescence), rather than uncontrolled
proliferation.

Ultimately, the growth-inhibitory pathways may prod the cells into apoptosis.


 

144

Another set of tumor suppressors seem to be involved in cell differentiation, causing cells to
enter a postmitotic, differentiated pool without replicative potential.

Similar to mitogenic signals,
growth-inhibitory, pro-differentiation signals originate outside the cell and use receptors, signal
transducers, and nuclear transcription regulators to accomplish their effects; tumor suppressors
form a portion of these networks.

145

TABLE 7-8 -- Selected Tumor Suppressor Genes Involved in Human Neoplasms

  • TGF-β receptor
  • E-cadherin
  • NF1
  • NF2
  • APC/β- catenin
  • PTEN
  • SMAD2 and SMAD4
  • RB1
  • p53
  • WT1
  • P16/INK4a
  • BRCA1 and BRCA2

146

TABLE 7-8 -- Selected Tumor Suppressor Genes Involved in Human Neoplasms

 

 

   Cell surface

  • TGF-β receptor
  • E- cadherin

147

TABLE 7-8 -- Selected Tumor Suppressor Genes Involved in Human Neoplasms

 

 

TGF-β
receptor

  • Function :  Growth inhibition
  • Tumors Associated : Carcinomas of colon
    with Somatic
    Mutations
  • Tumors Assocated: Unknown
    with Inherited
    Mutations

148

TABLE 7-8 -- Selected Tumor Suppressor Genes Involved in Human Neoplasms

 

 

Ecadherin

  • Function : Cell adhesion
  • Tumors Associated:Carcinoma of stomach
    with Somatic
    Mutations
  • Familial gastric cancer

149

TABLE 7-8 -- Selected Tumor Suppressor Genes Involved in Human Neoplasms

 

 

Inner aspect
of plasma
membrane

 

NF1

  • Function: Inhibition of RAS signal
    transduction and of
    p21 cell cycle inhibitor
  • Tumors Associated :Neuroblastomas
    with Somatic
    Mutations
  • Tumors Assocated : Neurofibromatosis type
    1 and sarcomas
    with Inherited
    Mutations

150

 

 

TABLE 7-8 -- Selected Tumor Suppressor Genes Involved in Human Neoplasms

 

Cytoskeleton :NF2

  • Function : Cytoskeletal stability
  • Tumors Associated : Schwannomas and
    meningiomas
    with Somatic
    Mutations
  • Tumors Assocated: Neurofibromastosis type
    2, acoustic
    schwannomas, and
    meningiomas
    with Inherited
    Mutations

151

TABLE 7-8 -- Selected Tumor Suppressor Genes Involved in Human Neoplasms

 

Cytosol

  • APC/β-
    catenin
  • PTEN
  • SMAD2
    and
    SMAD4

152

TABLE 7-8 -- Selected Tumor Suppressor Genes Involved in Human Neoplasms

 

Cytosol : APC/β-
catenin

  • Function : Inhibition of signal
    transduction
  • Tumors Associated with Somatic Mutations: Carcinomas of
    stomach, colon,
    pancreas; melanoma
  • Tumors Assocated with Inherited Mutations : Familial adenomatous
    polyposis coli/colon
    cancer

153

TABLE 7-8 -- Selected Tumor Suppressor Genes Involved in Human Neoplasms

 

Cytosol : PTEN

  • Function :PI3 kinase signal
    transduction
  • Tumors Associated with Somatic Mutations:
    • Endometrial and
      prostate cancers
  • Tumors Assocated with Inherited Mutations
    • Cowden syndrome

154

TABLE 7-8 -- Selected Tumor Suppressor Genes Involved in Human Neoplasms

 

Cytosol : SMAD2
and
SMAD4

  • Function :TGF-β signal
    transduction
  • Tumors Associated with Somatic
    Mutations:
    • Colon, pancreas tumors
  • Tumors Assocated with Inherited
    Mutations : Unknown

155

TABLE 7-8 -- Selected Tumor Suppressor Genes Involved in Human Neoplasms

 

Nucleus

  • RB1
  • p53
  • WT1
  • P16/INK4a
  • BRCA1 and BRCA2

156

TABLE 7-8 -- Selected Tumor Suppressor Genes Involved in Human Neoplasms

 

Nucleus : RB1

  • Function: Regulation of cell cycle
  • Tumors Associated with Somatic Mutations
    • Retinoblastoma;
      osteosarcoma
      carcinomas of breast,
      colon, lung
  • Tumors Assocated with Inherited Mutations
    • Retinoblastomas,
      osteosarcoma

157

TABLE 7-8 -- Selected Tumor Suppressor Genes Involved in Human Neoplasms

 

Nucleus : p53

  • Function : Cell cycle arrest and
    apoptosis in response
    to DNA damage
  • Tumors Associated with Somatic Mutations
    • Most human cancers
  • Tumors Assocated with Inherited Mutations
    • Li-Fraumeni syndrome;
      multiple carcinomas and
      sarcomas

158

TABLE 7-8 -- Selected Tumor Suppressor Genes Involved in Human Neoplasms

Nucleus : WT1

  • Function : Nuclear transcription
  • Tumors Associated with Somatic Mutations
    • Wilms' tumor
  • Tumors Assocated with Inherited Mutations
    • Wilms' tumor

159

TABLE 7-8 -- Selected Tumor Suppressor Genes Involved in Human Neoplasms

 

Nucleus : P16/INK4a

  • Function : Regulation of cell cycle
    by inhibition of
    cyclindependent
    kinases
  • Tumors Associated with Somatic Mutations:
    • Pancreatic, breast,
      and esophageal
      cancers
  • Tumors Assocated with Inherited Mutations
    • Malignant melanoma

160

TABLE 7-8 -- Selected Tumor Suppressor Genes Involved in Human Neoplasms

 

Nucleus : BRCA1 and BRCA2

  • Function : DNA repair
  • Tumors Associated with Somatic Mutations:
    • Unknown
  • Tumors Assocated with Inherited Mutations
    • Carcinomas of female
      breast and ovary;
      carcinomas of male
      breast

161

What is RB?

RB protein, the product of the RB gene, is a ubiquitously expressed nuclear phosphoprotein
that plays a key role in regulating the cell cycle.

162

RB exists in:

  •  an active hypophosphorylated 
    • state in quiescent cells and an
  • inactive hyperphosphorylated state
    • in the G1/S cell cycle transition

163

The importance of RB lies in its enforcement of :

 

 

G1, or the gap between
mitosis (M) and DNA replication (S).

 

 

164

In embryos, cell divisions proceed at an amazing clip, with DNA replication beginning immediately after mitosis ends. 

However, as development proceeds,
two gaps are incorporated into the cell cycle: 

 

  • Gap 1 (G1) between mitosis (M) and DNA replication (S), and
  • Gap 2 (G2) between DNA replication (S) and mitosis (M) (see Fig. 7-29 ).

165

Although each phase of the cell cycle circuitry is monitored carefully, the transition from ___________-
is believed to be an extremely important checkpoint in the cell cycle clock

G1 to S

 

 

Once cells cross the
G1 checkpoint they can pause the cell cycle for a time, but they are obligated to complete
mitosis.

 

 

166

In G1, however, cells can exit the cell cycle, either temporarily, called ____________

quiescence

167

In G1, however, cells can exit the cell cycle
permanently, called ____________

senescence. 

 

 

 

 

168

In G1, therefore, diverse signals are integrated to determine
whether the cell should enter the cell cycle, exit the cell cycle and differentiate, or die.

____________ is a
key node in this decision process.

To understand why RB is such a crucial player, we must
review the mechanisms that police the G1 phase.

RB

169

Q image thumb

FIGURE 7-31 The role of RB in regulating the G1-S checkpoint of the cell cycle.

 

Hypophosphorylated RB in complex with the E2F transcription factors binds to DNA, recruits
chromatin-remodeling factors (histone deacetylases and histone methyltransferases), and
inhibits transcription of genes whose products are required for the S phase of the cell cycle.

 

When RB is phosphorylated by the cyclin D–CDK4, cyclin D–CDK6, and cyclin E–CDK2
complexes, it releases E2F. The latter then activates transcription of S-phase genes. The
phosphorylation of RB is inhibited by CDKIs, because they inactivate cyclin-CDK complexes.
Virtually all cancer cells show dysregulation of the G1-S checkpoint as a result of mutation in
one of four genes that regulate the phosphorylation of RB; these genes are RB1, CDK4, the
genes encoding cyclin D proteins, and CDKN2A (p16). EGF, epidermal growth factor; PDGF,
platelet-derived growth factor; TGF-β, transforming growth factor-beta.

170

The initiation of DNA replication requires the activity of __________________________

  • cyclin E–CDK2 complexes,
  • and expression of cyclin E is dependent on the E2F family of transcription factors.

171

Early in G1, RB is
in its hypophosphorylated active form, and it binds to and inhibits the E2F family of transcription
factors, preventing transcription of cyclin E. Hypophosphorylated RB blocks E2F-mediated
transcription in at least two ways

  • First, it sequesters E2F, preventing it from interacting with other transcriptional activators.
  • Second, RB recruits chromatin-remodeling proteins, such as histone deacetylases and histone methyltransferases, which bind to the promoters of E2F-responsive genes such as cyclin E.

 

These enzymes modify chromatin so as
to make promoters insensitive to transcription factors.

 

172

What is cyclin D–CDK4/6 complexes. ?

Mitogenic signaling leads to cyclin D
expression and activation of cyclin D–CDK4/6 complexes. 

These complexes phosphorylate RB,
inactivating the protein and releasing E2F to induce target genes such as cyclin E. 

 

 

173

What is cyclin E?

Expression
of cyclin E then stimulates DNA replication and progression through the cell cycle.

 

 

174

What happens when cells enter the S phase?

When the
cells enter S phase, they are committed to divide without additional growth factor stimulation.


During the ensuing M phase the phosphate groups are removed from RB by cellular
phosphatases, regenerating the hypophosphorylated form of RB.

 

E2Fs are not the sole
effectors of Rb-mediated G1 arrest. Rb also controls the stability of the cell cycle inhibitor p27

175

If RB is absent (as a result of gene mutations) or its ability to regulate E2F transcription factors
is derailed,what happens then?

 the molecular brakes on the cell cycle are released, and the cells move through the
cell cycle.

 

 

176

What is an RB pocket?

​The mutations of RB genes found in tumors are localized to a region of the RB
protein, called the “RB pocket,” that is involved in binding to E2F.

 

However, the versatile RB
protein has also been shown to bind to a variety of other transcription factors that regulate cell
differentiation. [65]

 

For example, RB stimulates myocyte-, adipocyte-, melanocyte-, and
macrophage-specific transcription factors.

 

Thus, the RB pathway couples control of cell cycle
progression at G1 with differentiation, which may explain how differentiation is associated with
exit from the cell cycle. In addition to these dual activities, RB can also induce senescence,
discussed below

177

It was mentioned previously that germline loss or mutations of the RB gene predispose to
occurrence of retinoblastomas and to a lesser extent osteosarcomas. Furthermore, somatically
acquired RB mutations have been described in glioblastomas, small-cell carcinomas of lung,
breast cancers, and bladder carcinomas. Given the presence of RB in every cell and its
importance in cell cycle control, two questions arise:

 

(1) Why do patients with germline mutation
of the RB locus develop mainly retinoblastomas? (2) Why are inactivating mutations of RB not
much more common in human cancers?

The reason for the occurrence of tumors restricted to
the retina in persons who inherit one defective allele of RB is not fully understood, but some
possible explanations have emerged from the study of mice with targeted disruption of the rb
locus.

 

For instance, RB family members may partially complement its function in cell types other
than retinoblasts.

 

Indeed, RB is a member of a small family of proteins, so-called pocket proteins, which also include p107 and p130. [66]

All three proteins bind to E2F transcription
factors.

The complexity grows; there are seven E2F proteins (named E2F1 through E2F7),
which function as either transcriptional activators or repressors.

 

The pocket proteins are all
thought to regulate progression through the cell cycle as well as differentiation in a manner
similar to that described for RB above. However, each member of this protein family binds a
different set of E2F proteins and is also expressed at different times in the cell cycle. Thus,
although there is some redundancy in the network, their functions are not completely
overlapping. The complexity of the pocket protein–E2F network is just now being unraveled. For
example, in a mouse model of retinoblastoma, it has been shown that mutation of different
members of the network in various combinations generates retinoblastomas not just from
retinoblasts, but also from differentiated cells in the retina, such as horizontal interneurons

178

(2) Why are inactivating mutations of RB not
much more common in human cancers?

With respect to the second question (i.e., why the loss of RB is not more common in human
tumors), the answer is much simpler:

 

Mutations in other genes that control RB phosphorylation can mimic the effect of RB loss, and such genes are mutated in many cancers that may have normal RB genes.

 

Thus, for example, mutational activation of cyclin D or CDK4 would favor cell
proliferation by facilitating RB phosphorylation
.

As previously discussed, cyclin D is
overexpressed
in many tumors because of gene amplification or translocation.

Mutational
inactivation of CDKIs would also drive the cell cycle by unregulated activation of cyclins and
CDKs.

 

Thus, the emerging paradigm is that loss of normal cell cycle control is central to
malignant transformation and that at least one of four key regulators of the cell cycle
( p16/INK4a, cyclin D, CDK4, RB) is dysregulated in the vast majority of human cancers . [68] In
cells that harbor mutations in any one of these other genes, the function of RB is disrupted
even if the RB gene itself is not mutated

179

The transforming proteins of several oncogenic animal and human DNA viruses seem to act, in
part, how?

by neutralizing the growth-inhibitory activities of RB.

In these cases, RB protein is
functionally inactivated by the binding of a viral protein and no longer acts as a cell cycle
inhibitor.

Simian virus 40 and polyomavirus large T antigens, adenoviruses EIA protein, and
HPV E7 protein all bind to the hypophosphorylated form of RB.

The binding occurs in the same
RB pocket that normally sequesters E2F transcription factors; in the case of HPV the binding is
particularly strong for viral types, such as HPV type 16, that confer high risk for the
development of cervical carcinomas.

Thus, the RB protein, unable to bind the E2F transcription factors, is functionally inactivated, and the transcription factors are free to cause cell cycle
progression.

180

What is p53?

p53: Guardian of the Genome.

181

Where is gene p53 located?

The p53 gene is located on chromosome 17p13.1, and it is the most common target for genetic
alteration in human tumors

 

(The official name of the gene is TP53 and the protein is p53;
for the sake of simplicity, we refer to both as “p53”.) A little over 50% of human tumors contain
mutations in this gene. Homozygous loss of p53 occurs in virtually every type of cancer,
including carcinomas of the lung, colon, and breast—the three leading causes of cancer death.

182

In most cases, the inactivating mutations affect both p53 alleles and are acquired in somatic
cells
(not inherited in the germ line).

Less commonly, some individuals inherit one mutant p53
allele.

 

 

183

As with the RB gene, inheritance of one mutant allele predisposes individuals to develop
malignant tumors because only one additional “hit” is needed to inactivate the second, normal
allele.

Such individuals, said to have the __________, have a 25-fold greater chance
of developing a malignant tumor by age 50 than the general population. [70]

 

Li-Fraumeni syndrome

184

In contrast to
individuals who inherit a mutant RB allele, the spectrum of tumors that develop in persons with
the Li-Fraumeni syndrome is quite varied; the most common types of tumors are ________ 

 

As compared with
sporadic tumors, those that afflict patients with the Li-Fraumeni syndrome occur at a younger
age, and a given individual may develop multiple primary tumors

sarcomas,
breast cancer, leukemia, brain tumors, and carcinomas of the adrenal cortex.

185

What is the funciton of p53

The fact that p53 mutations are common in a variety of human tumors suggests that the p53
protein functions as a critical gatekeeper against the formation of cancer

 

Indeed, it is evident
that p53 acts as a “molecular policeman” that prevents the propagation of genetically damaged
cells.

p53 is a transcription factor that is at the center of a large network of signals that sense
cellular stress, such as DNA damag
e, shortened telomeres, and hypoxia.

Many activities of the
p53 protein are related to its function as a transcription factor.

 

Several hundred genes have
been shown to be regulated by p53 in numerous different contexts, but which genes are the key
for the p53 response is not yet clear.

 

Approximately 80% of the p53 point mutations present inhuman cancers are located in the DNA-binding domain of the protein.

 

However, the effects of
different point mutations vary considerably; in some cases there is complete abrogation of
transcriptional capabilities, whereas other mutants retain the ability to bind to and activate a
subset of genes. In addition to somatic and inherited mutations, p53 functions can be
inactivated by other mechanisms. As with RB, the transforming proteins of several DNA viruses,
including the E6 protein of HPV, can bind to and promote the degradation of p53. Also,
analagous to RB, it is thought that in the majority of tumors without a p53 mutation, the function
of the p53 pathway is blocked by mutation in another gene that regulates p53 function. For
example, MDM2 and MDMX stimulate the degradation of p53; these proteins are frequently
overexpressed in malignancies in which the gene encoding p53 is not mutated. Indeed, MDM2
is amplified in 33% of human sarcomas, thereby causing functional loss of p53 in these
tumors

 

186

p53 thwarts neoplastic transformation by three interlocking mechanisms: 

  • activation of temporary cell cycle arrest (quiescence),
  • induction of permanent cell cycle arrest (senescence),
  • or triggering of programmed cell death (apoptosis).

187

In nonstressed, healthy cells, p53 has a short half-life (20 minutes), why?

because of its association
with MDM2, a protein that targets it for destruction.

188

When the cell is stressed, for example by an
assault on its DNA, what happens to p53?

p53 undergoes post-transcriptional modifications that release it from MDM2
and increase its half-life.

 

Unshackled from MDM2, p53 also becomes activated as a
transcription factor.

 

189

Hundreds of genes whose transcription is triggered by p53 have been found. [74,] [75] 

 

They can be grouped into two broad categories: 

  • those that cause cell cycle arrest and those that cause apoptosis. If DNA damage can be repaired during cell cycle arrest, the cell reverts to a normal state;
  • if the repair fails, p53 induces apoptosis or senescence.

Recently, however, the plot has thickened. It has been known that repression of a subset of proproliferative
and anti-apoptotic genes is key to the p53 response, but it was not clear how p53
achieved repression, since in most assays it seemed to be an activator of transcription.

 

At this
point enter the recently famous miRNAs, the small guys with big clubs. It has been shown that
p53 activates transcription of the mir34 family of miRNAs (mir34a–mir34c). [76]

 

miRNAs, as
discussed in Chapter 5 , bind to cognate sequences in the 3′ untranslated region of mRNAs,
preventing translation ( Fig. 7-32B ).

 

Interestingly, blocking mir34 severely hampered the p53
response in cells, while ectopic expression of mir34 without p53 activation is sufficient to induce
growth arrest and apoptosis. Thus, mir34 microRNAs are able to recapitulate many of the
functions of p53 and are necessary for these functions, demonstrating the importance of mir34
to the p53 response. Targets of mir34s include pro-proliferative genes such as cyclins, and
anti-apoptotic genes such as BCL2. p53 regulation of mir34 explains, at least in part, how p53
is able to repress gene expression, and it seems that regulation of this miRNA is crucial for the
p53 response.

190

Q image thumb

FIGURE 7-32

 

A, The role of p53 in maintaining the integrity of the genome. Activation of normal p53 by DNA-damaging agents or by hypoxia leads to cell cycle arrest in G1 and induction of DNA repair, by transcriptional up-regulation of the cyclin-dependent kinase
inhibitor CDKN1A (p21) and the GADD45 genes. Successful repair of DNA allows cells to
proceed with the cell cycle; if DNA repair fails, p53 triggers either apoptosis or senescence.
In cells with loss or mutations of p53, DNA damage does not induce cell cycle arrest or DNA
repair, and genetically damaged cells proliferate, giving rise eventually to malignant
neoplasms.

B, p53 mediates gene repression by activating transcription of miRNAs. p53
activates transcription of the mir34 family of miRNAs. mir34s repress translation of both
proliferative genes, such as cyclins, and anti-apoptotic genes, such as BCL2.

Repression of
these genes can promote either quiescence or senescence as well as apoptosis.

191

The manner in which p53 senses DNA damage and determines the adequacy of DNA repair is
beginning to be understood.

 

The key initiators of the DNA-damage pathway are two related
protein kinases: 

  • ataxia-telangiectasia mutated (ATM) and  ataxia-telangiectasia and
  • Rad3 related (ATR). [77,] [78]

192

What is ATM gene?

 As the name implies, the ATM gene was originally identified as the germ-line mutation in individuals with ataxia-telangiectasia.

 

Persons with this disease, which is characterized by an inability to repair certain kinds of DNA damage, suffer from an increased incidence of cancer.

 

The types of damage sensed by ATM and ATR are different, but the
downstream pathways they activate are similar. Once triggered, both ATM and ATR phosphorylate a variety of targets, including p53 and DNA-repair proteins. Phosphorylation of these two targets leads to a pause in the cell cycle and stimulation of DNA-repair pathways, respectively.

193

p53-mediated cell cycle arrest may be considered the primordial response to DNA damage (
Fig. 7-32 )

 

. It occurs late in the G1 phase and is caused mainly by p53-dependent transcription
of the CDK inhibitor

(p21).

As discussed, p21 inhibits cyclin-CDK complexes and
phosphorylation of RB
, thereby preventing cells from entering G1 phase.

 

Such a pause in cell
cycling is welcome, because it gives the cells “breathing time” to repair DNA damage. p53 also
helps the process by inducing certain proteins
, such as GADD45 (growth arrest and DNA
damage),
that help in DNA repair. [75]

 

p53 can stimulate DNA-repair pathways by transcriptionindependent
mechanisms as well.

 

If DNA damage is repaired successfully, p53 up-regulates
transcription of MDM2, leading to its own destruction and thus releasing the cell cycle block. If
the damage cannot be repaired, the cell may enter p53-induced senescence or undergo p53-
directed apoptosis.

194

p53-induced senescence is a permanent cell cycle arrest characterized by:

by specific changes in
morphology and gene expression that differentiate it from quiescence or reversible cell cycle
arrest.

195

What requirement for senescence?

 Senescence requires activation of p53 and/or RB and expression of their mediators,
such as the CDK inhibitors, and is generally irreversible, although it may require the continued
expression of p53.

 

The mechanisms of senescence are unclear but involve epigenetic changes
that result in the formation of heterochromatin at different loci throughout the genome.

 

These senescence-associated heterochromatin foci include pro-proliferative genes regulated
by E2F; this drastically and permanently alters expression of these E2F targets. Like all p53
responses, senescence may be stimulated in response to a variety of stresses, such as
unopposed oncogene signaling, hypoxia, and shortened telomeres.

196

What is the ultimate protective mechanism against neoplastic transformation?

p53-induced apoptosis of cells with irreversible DNA damage is the ultimate protective
mechanism against neoplastic transformation.

 

p53 directs the transcription of several proapoptotic
genes such as BAX and PUMA (approved name BBC3; described later).

 

Exactly how
a cell decides whether to repair its DNA or to enter apoptosis is unclear.

 

It appears that the
affinity of p53 for the promoters and enhancers of DNA-repair genes is stronger than its affinity
for pro-apoptotic genes. [80]

 

Thus, the DNA-repair pathway is stimulated first, while p53 continues to accumulate.

Eventually, if the DNA damage is not repaired, enough p53 accumulates to stimulate transcription of the pro-apoptotic genes and the cell dies.

While this
scheme is generally correct, there seem to be important cell type–specific responses as well,
with some cell types succumbing to apoptosis early, while others opt for senescence. [80]

Such
differential responses may be related to the functions of other p53 family members expressed in
different cell types (see below).

197

To summarize, p53 links cell damage with DNA repair, cell cycle arrest, and apoptosis.

 

In response to DNA damage, p53 is phosphorylated by genes that sense the damage and are
involved in DNA repair. p53 assists in DNA repair by causing G1 arrest and inducing DNArepair
genes.

A cell with damaged DNA that cannot be repaired is directed by p53 to undergo apoptosis (see Fig. 7-32 ). In view of these activities, p53 has been rightfully called a “guardian of the genome.

 

” With loss of function of p53, DNA damage goes unrepaired, mutations
accumulate in dividing cells, and the cell marches along a one-way street leading to malignant
transformation

198

The ability of p53 to control apoptosis in response to DNA damage has important practical
therapeutic implications. Irradiation and chemotherapy, the two common modalities of cancer
treatment, mediate their effects by inducing DNA damage and subsequent apoptosis.

 

Tumors
that retain normal p53 are more likely to respond to such therapy than tumors that carry
mutated alleles of the gene.

 

Such is the case with testicular teratocarcinomas and childhood
acute lymphoblastic leukemias. By contrast, tumors such as lung cancers and colorectal
cancers, which frequently carry p53 mutations, are relatively resistant to chemotherapy and
irradiation.

 

Various therapeutic modalities aimed at increasing normal p53 activity in tumor cells
that retain this type of activity or selectively killing cells with defective p53 function are being
investigated.

199

The discovery of p53 family members p63 and p73 has revealed that p53 has collaborators.

Indeed, p53, p63, and p73 are players in a complex network with significant cross-talk that is

only beginning to be unraveled. [81,] [82] p53 is ubiquitously expressed, while p63 and p73

show more tissue specificity. For example, p63 is essential for the differentiation of stratified

squamous epithelia, while p73 has strong pro-apoptotic effects after DNA damage induced by

chemotheraputic agents. Furthermore, both p63 and p73, and probably p53 as well, are

expressed as different isoforms, some of which act as transcriptional activators and others that

function as dominant negatives. An illustrative example of the concerted actions of these three

musketeers is seen in the so-called basal subset of breast cancers, which have a poor

prognosis. These tumors have been shown to have mutations in p53 and additionally express a

dominant-negative version of p63 that antagonizes the apoptotic activity of p73. This

perturbation of the p53p63-p73 network contributes to the chemoresistance and poor

prognosis of these tumors

200

What is APC?

Adenomatous polyposis coli genes (APC) represents a class of tumor suppressors whose main
function is to down-regulate growth-promoting signals.

201

Germ-line mutations at the APC (5q21)
loci are associated with familial adenomatous polyposis, in which all individuals born with one
mutant allele develop thousands of adenomatous polyps in the colon during their teens or 20s

(familial adenomatous polyposis; Chapter 17 ).

 

Almost invariably, one or more of these polyps
undergoes malignant transformation, giving rise to colon cancer.

 

As with other tumor
suppressor genes, both copies of the APC gene must be lost for a tumor to arise

. This
conclusion is supported by the development of colon adenomas in mice with targeted disruption
of apc genes in the colonic mucosa. [84]

 

As discussed later, several additional mutations must
occur if cancers are to develop in the adenomas. In addition to these tumors, which have a
strong hereditary predisposition, 70% to 80% of nonfamilial colorectal carcinomas and sporadic
adenomas also show homozygous loss of the APC gene, thus firmly implicating APC loss in the
pathogenesis of colonic tumors

202

What is the funciton of APC?

APC is a component of the WNT signaling pathway, which has a major role in controlling cell
fate, adhesion, and cell polarity during embryonic development ( Fig. 7-33 ).

 

WNT signaling is
also required for self-renewal of hematopoietic stem cells.

 

WNT signals through a family of cell
surface receptors called frizzled (FRZ), and stimulates several pathways, the central one
involving β-catenin and APC.

203

Q image thumb

FIGURE 7-33 A, The role of APC in regulating the stability and function of β-catenin. APC
and β-catenin are components of the WNT signaling pathway. In resting cells (not exposed to
WNT), β-catenin forms a macromolecular complex containing the APC protein. This complex
leads to the destruction of β-catenin, and intracellular levels of β-catenin are low. B, When
cells are stimulated by WNT molecules, the destruction complex is deactivated, β-catenin
degradation does not occur, and cytoplasmic levels increase. β-catenin translocates to the
nucleus, where it binds to TCF, a transcription factor that activates genes involved in cell
cycle progression. C, When APC is mutated or absent, the destruction of β-catenin cannot
occur. β-catenin translocates to the nucleus and coactivates genes that promote entry into
the cell cycle, and cells behave as if they are under constant stimulation by the WNT
pathway.

204

An important function of the APC protein is to down-regulate β-catenin. Why?

 In the absence of WNT
signaling APC causes degradation of β-catenin, preventing its accumulation in the
cytoplasm
. [85]

 

It does so by forming a macromolecular complex with β-catenin, axin, and
GSK3β
, which leads to the phosphorylation and eventually ubiquitination of β-catenin and
destruction by the proteasome.

 

Signaling by WNT blocks the APC-AXIN-GSK3β destruction
complex, allowing β-catenin to translocate from the cytoplasm to the nucleus.

 

 In the cell
nucleus, β-catenin forms a complex with TCF, a transcription factor that up-regulates cellular
proliferation by increasing the transcription of c-MYC, cyclin D1, and other genes.

 

Since
inactivation of the APC gene disrupts the destruction complex, β-catenin survives and
translocates to the nucleus, where it can activate transcription in cooperation with TCF. [85]

 

Thus, cells with loss of APC behave as if they are under continuous WNT signaling.

 

The
importance of the APC/β-catenin signaling pathway in tumorigenesis is attested to by the fact
that colon tumors that have normal APC genes harbor mutations in β-catenin that prevent its
destruction by APC, allowing the mutant protein to accumulate in the nucleus. Dysregulation of
the APC/β-catenin pathway is not restricted to colon cancers; mutations in the β-catenin gene
are present in more than 50% of hepatoblastomas and in approximately 20% of hepatocellular
carcinomas.

205

What is the function of β-catenin? 

β-catenin binds to the cytoplasmic tail of Ecadherin,
a cell surface protein that maintains intercellular adhesiveness

 

. Loss of cell-cell
contact, such as in a wound or injury to the epithelium, disrupts the interaction between Ecadherin
and β-catenin, and allows β-catenin to travel to the nucleus and stimulate proliferation;
this is an appropriate response to injury that can help repair the wound.

 

Re-establishment of
these E-cadherin contacts as the wound heals leads to β-catenin again being sequestered at
the membrane and reduction in the proliferative signal; these cells are said to be “contactinhibited.”
 

 

Loss of contact inhibition, by mutation of the E-cadherin/β-catenin axis, or by other
methods, is a key characteristic of carcinomas.

 

Furthermore, loss of cadherins can favor the
malignant phenotype by allowing easy disaggregation of cells, which can then invade locally or
metastasize. Reduced cell surface expression of E-cadherin has been noted in many types of
cancers, including those that arise in the esophagus, colon, breast, ovary, and prostate. [87]
Germline mutations of the E-cadherin gene can predispose to familial gastric carcinoma, and
mutation of the gene and decreased E-cadherin expression are present in a variable proportion
of gastric cancers of the diffuse type. The molecular basis of reduced E-cadherin expression is
varied. In a small proportion of cases, there are mutations in the E-cadherin gene (located on
16q); in other cancers, E-cadherin expression is reduced as a secondary effect of mutations in
β-catenin genes. Additionally, E-cadherin may be down-regulated by transcription repressors,
such as SNAIL, which have been implicated in epithelial-to-mesenchymal transition and
metastasis

206

There is little doubt that many more tumor suppressor genes remain to be discovered. Often,
their location is suspected by the detection of consistent sites of chromosomal deletions or by
analysis of LOH. Some of the tumor suppressor genes that are associated with well-defined
clinical syndromes are briefly described below (see Table 7-8 ):

207

What is INK4a/ARF?

Also called the CDKN2A gene locus, the INK4a/ARF locus encodes two protein products;

the
p16/INK4a CDKI, which blocks cyclin D/CDK2-mediated phosphorylation of RB, keeping the RB
checkpoint in place
.

The second gene product, p14/ARF, activates the p53 pathway by inhibiting MDM2 and preventing destruction of p53

 

. Both protein products function as tumor
suppressors, and thus mutation or silencing of this locus impacts both the RB and p53
pathways.

p16 in particular is crucial for the induction of senescence.

 

Mutations at this locus
have been detected in bladder, head and neck tumors, acute lymphoblastic leukemias, and
cholangiocarcinomas.

In some tumors, such as cervical cancer, p16/INK4a is frequently silenced
by hypermethylation of the gene, without the presence of a mutation (see discussion of
epigenetic changes).

 

The other CDKIs also function as tumor suppressors and are frequently
mutated or otherwise silenced in many human malignancies, including 20% of familial
melanomas, 50% of sporadic pancreatic adenocarcinomas, and squamous cell carcinomas of
the esophagus.

208

What is The TGF-β Pathway? 

In most normal epithelial, endothelial, and hematopoietic cells, TGF-β is a potent inhibitor of
proliferation.

 

It regulates cellular processes by binding to a serine-threonine kinase complex composed of TGF-β receptors I and II.

Dimerization of the receptor upon ligand binding leads to activation of the kinase and phosphorylation of receptor SMADs (R-SMADs).

 

Upon phosphorylation, R-SMADs can enter the nucleus, bind to SMAD-4, and activate transcription of
genes, including the CDKIs p21 and p15/INK4b. In addition, TGF-β signaling leads to
repression of c-MYC, CDK2, CDK4, and cyclins A and E.

 

As can be inferred from our earlier
discussion, these changes result in decreased phosphorylation of RB and cell cycle arrest.

209

In many forms of cancer the growth-inhibiting effects of TGF-β pathways are impaired by
mutations in the TGF-β signaling pathway. 

These may affect the?

These mutations may affect the type II TGF-β
receptor or interfere with SMAD molecules that serve to transduce antiproliferative signals from
the receptor to the nucleus.

 

210

Mutations affecting the type II receptor are seen in cancers of the
 

 

colon, stomach, and endometrium. 

211

Mutational inactivation of SMAD4 is common in 

pancreatic cancers.

 

In 100% of pancreatic cancers and 83% of colon cancers, at least one component of
the TGF-β pathway is mutated .

 

However, in many cancers, loss of TGF-β-mediated growth
inhibition occurs at a level downstream of the core signaling pathway, for example, loss of p21
and/or persistent expression of c-Myc.

 

These tumor cells can then use other elements of the
TGFβ–induced program, including immune system suppression/evasion or promotion of
angiogenesis, to facilitate tumor progression. [89] Thus TGF-β can function to prevent or
promote tumor growth, depending on the state of other genes in the cell.

212

What is PTEN?

PTEN (Phosphatase and tensin homologue) is a membrane-associated phosphatase encoded
by a gene on chromosome 10q23
that is mutated in

213

What is cowden syndrome?

Cowden syndrome, an autosomal dominant
disorder marked by frequent benign growths, such as tumors of the skin appendages, and an
increased incidence of epithelial cancers, particularly of the breast
( Chapter 23 ), endometrium, and thyroid.

 

 

214

How does PTEN act as tumor suppressor?

PTEN acts as a tumor suppressor by serving as a brake on the prosurvival/
pro-growth PI3K/AKT pathway

215

What is the PI3K/AKT pathway?

As you will recall from Chapter 3 , this pathway
is normally stimulated (along with the RAS and JAK/STAT pathways) when ligands bind to
receptor tyrosine kinases and involves a cascade of phosphorylation events.

 

First, PI3K
(phosphoinositide 3-kinase) phosphorylates the lipid inositide-3-phosphate to give rise to inositide-3,4,5-triphosphate, which binds and activates the kinase

PDK1. PDK1 and other
factors in turn phosphorylate and activate the serine/threonine kinase AKT, which is a major
node in the pathway with several important functions.

 

By phosphorylating a number of
substrates, including BAD and MDM2, AKT enhances cell survival.

 

 

216

AKT also inactivates the
TSC1/TSC2 complex.

What is  TSC1 and TSC2? 

 TSC1 and TSC2 are the products of two tumor suppressor genes that
are mutated in tuberous sclerosis ( Chapter 28 ), an autosomal dominant disorder associated
with developmental malformations and unusual benign neoplasms such as cardiac
rhabdomyomas ( Chapter 12 ), renal angiomyolipomas, and giant cell astrocytomas.

 

 

217

What tuberous sclerosis?

 tuberous sclerosis ( Chapter 28 ), an autosomal dominant disorder associated
with developmental malformations and unusual benign neoplasms such as cardiac
rhabdomyomas ( Chapter 12 ), renal angiomyolipomas, and giant cell astrocytomas

218

. Inactivation
of TSC1/TSC2 unleashes the activity of yet another kinase called mTOR (mammalian target of
rapamycin, a potent immunosuppressive drug),
which stimulates the uptake of nutrients such as
glucose and amino acids that are needed for growth and augments the activity of several
factors that are required for protein synthesis.

 

 

219

What is the most commonly mutated pathway in human cancer ?

Although acquired loss of PTEN function is one
of the most common ways that PI3K/AKT signaling is upregulated in various cancers, many
other components of the pathway, including PI3K itself, may also be mutated so as to increase
signaling. 

 

Considering all of these molecular lesions collectively, it is said that this may be the
most commonly mutated pathway in human cancer.

As a result there is great interest in
targeting the PI3K/AKT pathway with inhibitors of mTOR, AKT, and other kinases in the pathway.

220

What is NF1?

Individuals who inherit one mutant allele of the NF1 gene develop numerous benign
neurofibromas and optic nerve gliomas as a result of inactivation of the second copy of the
gene. [92]

 

This condition is called neurofibromatosis type 1 ( Chapter 27 ).

Some of the
neurofibromas later develop into malignant peripheral nerve sheath tumors.

 

Neurofibromin, the
protein product of the NF1 gene, contains a GTPase-activating domain, which regulates signal
transduction through RAS proteins.

 

Recall that RAS transmits growth-promoting signals and
flips back and forth between GDP-binding (inactive) and GTP-binding (active) states.
Neurofibromin facilitates conversion of RAS from an active to an inactive state.

With loss of
neurofibromin function, RAS is trapped in an active, signal-emitting state.

221

What is NF2?

Germline mutations in the NF2 gene predispose to the development of neurofibromatosis type
2. [93]

 

As discussed in Chapter 27 , individuals with mutations in NF2 develop benign bilateral
schwannomas of the acoustic nerve
.

 

In addition, somatic mutations affecting both alleles of NF2
have also been found in sporadic meningiomas and ependymomas.

 

 

222

What is the product of NF2 gene?

The product of the NF2
gene, called neurofibromin 2 or merlin, shows a great deal of homology with the red cell
membrane cytoskeletal protein
4.1 ( Chapter 14 ), and is related to the ERM (ezrin, radixin, and
moesin)
family of membrane cytoskeleton-associated proteins.

 

Although the mechanism by
which merlin deficiency leads to carcinogenesis is not known, cells lacking this protein are not
capable of establishing stable cell-to-cell junctions and are insensitive to normal growth arrest
signals generated by cell-to-cell contact.

Merlin is a key member of the Salvador-

Warts-Hippo
(SWH) tumor suppressor pathway, originally described in Drosophila.

 

The signaling pathway
controls organ size by modulating cell growth, proliferation, and apoptosis. Many human
homologues of genes in the SWH pathway have been implicated in human cancers.

223

What is VHL?

Germline mutations of the von Hippel-Lindau (VHL) gene on chromosome 3p are associated
with hereditary renal cell cancers, pheochromocytomas, hemangioblastomas of the central nervous system, retinal angiomas, and renal cysts. [60]

 

Mutations of the VHL gene have also
been noted in sporadic renal cell cancers ( Chapter 20 ).

The VHL protein is part of a ubiquitin
ligase complex. A critical substrate for this activity is HIF1α (hypoxia-inducible transcription
factor 1α)
.

 

In the presence of oxygen, HIF1α is hydroxylated and binds to the VHL protein,
leading to ubiquitination and proteasomal degradation.

 

This hydroxylation reaction requires
oxygen; in hypoxic environments the reaction cannot occur, and HIF1α escapes recognition by
VHL and subsequent degradation.

 

HIF1α can then translocate to the nucleus and turn on many
genes, such as the growth/angiogenic factors vascular endothelial growth factor (VEGF) and
PDGF.

 

Lack of VHL activity prevents ubiquitination and degradation of HIF1α and is associated
with increased levels of angiogenic growth factors.

224

What is WT1?

The WT1 gene, located on chromosome 11p13, is associated with the development of Wilms'
tumor, a pediatric kidney cancer. [95]

 

Both inherited and sporadic forms of Wilms' tumor occur, and mutational inactivation of the WT1 locus has been seen in both forms.

The WT1 protein is
a transcriptional activator of genes involved in renal and gonadal differentiation.

 

It regulates the
mesenchymal-to-epithelial transition
that occurs in kidney development.

 

Though not precisely
known, it is likely that the tumorigenic effect of WT1 deficiency is intimately connected with the
role of the gene in the differentiation of genitourinary tissues
.

 

Interestingly, although WT1 is a
tumor suppressor in Wilms' tumor, a variety of adult cancers, including leukemias and breast
carcinomas
, have also been shown to overexpress WT1.

 

Since these tissues do not normally
express WT1 at all, it has been suggested that WT1 may function as an oncogene in these
cancers.

 

Another Wilms' gene, WT2, located on 11p15, is associated with the Beckwith-
Wiedemann syndrome ( Chapter 10 ).

225

What is PTCH1 and PTCH2

PTCH1 and PTCH2 are tumor suppressor genes that encode a cell membrane protein
(PATCHED),
which functions as a receptor for a family of proteins called Hedgehog. [96]

 

The
Hedgehog/PATCHED pathway regulates several genes, including TGF-β and PDGFRA and
PDGFRB.

 

226

What is Gorlin syndrome?

Mutations in PTCH are related to Gorlin syndrome, an inherited condition also known
as nevoid basal cell carcinoma syndrome (see Chapter 26 ).

PTCH mutations are present in
20% to 50% of sporadic cases of basal cell carcinoma.

About one half of such mutations are of
the type caused by UV exposure.

227

Accumulation of neoplastic cells may result not only from activation of growth-promoting
oncogenes or inactivation of growth-suppressing tumor suppressor genes, but also from
mutations in the genes that regulate apoptosis. [97] [98] [99]

Thus, apoptosis represents a
barrier that must be surmounted for cancer to occur.

 

In the adult, cell death by apoptosis is a
physiologic response to several pathologic conditions that might contribute to malignancy if the
cells remained viable.

 

A cell with genomic injury can be induced to die, preventing the
accumulation of cells with mutations.

 

 

228

A variety of signals, ranging from DNA damage to loss of dhesion to the basement membrane (termed anoikis), can trigger apoptosis.

 

A large family of
genes that regulate apoptosis has been identified. Before we can understand how tumor cells evade apoptosis, it is essential to review briefly the biochemical pathways to apoptosis.

229

there are two distinct programs that activate apoptosis, 

  • the extrinsic and
  • intrinsic pathways.

230

the sequence of events that lead
to apoptosis by signaling through the:

  •  death receptor CD95/Fas (extrinsic pathway) and by
  • DNAdamage (intrinsic pathway).

231

How is the extrinsic pathway initiated?

The extrinsic pathway is initiated when CD95/Fas binds to its
ligand, CD95L/FasL
, leading to trimerization of the receptor and its cytoplasmic death domains,
which attract the intracellular adaptor protein FADD.

 

This protein recruits procaspase 8 to form
the death-inducing signaling complex.

 

Procaspase 8 is activated by cleavage into smaller
subunits, generating caspase 8.

 

Caspase 8 then activates downstream caspases such as
caspase 3, a typical executioner caspase that cleaves DNA and other substrates to cause cell
death.
Additionally, caspase 8 can cleave and activate the BH3-only protein BID, activating the
intrinsic pathway as well.

232

What triggers the intrinsic pathway?

The intrinsic pathway of apoptosis is triggered by a variety of stimuli, including withdrawal of survival factors, stress, and injury.

 

Activation of this pathway leads to
permeabilization of the mitochondrial outer membrane, with resultant release of molecules, such
as cytochrome c, that initiate apoptosis.

 

 

233

The integrity of the mitochondrial outer membrane is
regulated by pro-apoptotic and anti-apoptotic members of the ________. [100]
 

BCL2 family of proteins

234

The pro-apoptotic proteins _are required for apoptosis and directly promote
mitochondrial permeabilization.

BAX and BAK 

235

Their action is inhibited by the anti-apoptotic members of this family exemplified by ________ 

BCL2 and BCL-XL.

236

A third set of proteins (so-called BH3-only proteins),
including_____________, regulate the balance between the pro- and anti-apoptotic
members of the BCL2 family.

 

 

 BAD, BID, and PUMA

 

The BH3-only proteins sense death-inducing stimuli and promote apoptosis by neutralizing the actions of anti-apoptotic proteins like BCL2 and BCL-XL.

 

When
the sum total of all BH3 proteins expressed “overwhelms” the anti-apoptotic BCL2/BCL-XL
protein barrier, BAX and BAK are activated and form pores in the mitochondrial membrane.

 


Cytochrome c leaks into the cytosol, where it binds to APAF1, activating caspase 9.

 

Like
caspase 8 of the extrinsic pathway, caspase 9 can cleave and activate the executioner
caspases.

 

The caspases can be inhibited by a family of proteins called Inhibitors of Apoptosis
Proteins (IAPs).

 

Some tumors avoid apoptosis by upregulating these proteins, and there is
interest in developing drugs that can block the interaction between IAPs and caspases.
Because of the pro-apoptotic effect of BH3-only proteins, efforts are underway to develop BH3
mimetic drugs

237

Q image thumb

FIGURE 7-34 CD95 receptor–induced and DNA damage–triggered pathways of apoptosis
and mechanisms used by tumor cells to evade cell death. (1) Reduced CD95 level. (2)
Inactivation of death-induced signaling complex by FLICE protein (caspase 8; apoptosis- related cysteine peptidase). (3) Reduced egress of cytochrome c from mitochondrion as a
result of up-regulation of BCL2. (4) Reduced levels of pro-apoptotic BAX resulting from loss
of p53. (5) Loss of apoptotic peptidase activating factor 1 (APAF1). (6) Up-regulation of
inhibitors of apoptosis (IAP). FADD, Fas-associated via death domain.

238

Within this framework it is possible to illustrate the multiple sites at which apoptosis is frustrated
by cancer cells [101] (see Fig. 7-34 ).

 

Starting from the surface, reduced levels of CD95/Fas
may render the tumor cells less susceptible to apoptosis by CD95L/FasL.

 

Some tumors have
high levels of FLIP, a protein that can bind death-inducing signaling complex and prevent
activation of caspase 8. Of all these genes, perhaps best established is the role of BCL2 in
protecting tumor cells from apoptosis. As discussed later, approximately 85% of B-cell
lymphomas of the follicular type ( Chapter 13 ) carry a characteristic t(14;18)(q32;q21)
translocation. Recall that 14q32, the site where immunoglobulin heavy-chain (IgH) genes are
found, is also involved in the pathogenesis of Burkitt lymphoma. Juxtaposition of this
transcriptionally active locus with BCL2 (located at 18q21) causes overexpression of the BCL2
protein. This in turn increases the BCL2/BCL-XL buffer, protecting lymphocytes from apoptosis
and allowing them to survive for long periods; there is therefore a steady accumulation of B
lymphocytes, resulting in lymphadenopathy and marrow infiltration. Because BCL2-
overexpressing lymphomas arise in large part from reduced cell death rather than explosive cell
proliferation, they tend to be indolent (slow growing) compared with many other lymphomas.

239

As mentioned before, p53 is an important pro-apoptotic gene that induces apoptosis in cells
that are unable to repair DNA damage.

 

The actions of p53 are mediated in part by
transcriptional activation of BAX, but there are other connections as well between p53 and the
apoptotic machinery. Thus, the apoptotic machinery in cancer may be thwarted by mutations
affecting the component proteins directly, as well as by loss of sensors of genomic integrity
such as p53.

240

LIMITLESS REPLICATIVE POTENTIAL: 

TELOMERASE

241

Explain the phenomenon of progressive shortening of telomeres at the end of choromosome?

As was discussed in the section on cellular aging ( Chapter 1 ), most normal human cells have
a capacity of 60 to 70 doublings.

After this, the cells lose their ability to divide and become
senescent. This phenomenon has been ascribed to progressive shortening of telomeres at the
ends of chromosomes

242

Indeed, short telomeres seem to be recognized by the DNA-repair machinery as double-stranded DNA breaks, and this leads to cell cycle arrest mediated by ______________ [102]

p53 and RB.

 

 

 

In cells in which the checkpoints are disabled by p53 or RB1 mutations, the nonhomologous end-joining pathway is activated as a last-ditch effort to save the cell, joining the shortened ends of two chromosomes. [103]

 

This inappropriately activated repair system
results in dicentric chromosomes that are pulled apart at anaphase, resulting in new doublestranded
DNA breaks.

 

The resulting genomic instability from the repeated bridge-fusionbreakage cycles eventually produces mitotic catastrophe, characterized by massive cell death.
It follows that for tumors to grow indefinitely, as they often do, loss of growth restraints is not
enough. Tumor cells must also develop ways to avoid both cellular senescence and mitotic
catastrophe ( Fig. 7-35 )

243

If during crisis a cell manages to reactivate telomerase, the bridgefusion-
breakage cycles cease and the cell is able to avoid death.

However, during the period of
genomic instability that precedes telomerase activation, numerous mutations could accumulate,
helping the cell march toward malignancy.

 

Passage through a period of genomic instability may
explain the complex karyotypes frequently seen in human carcinomas.

 

Telomerase, active in
normal stem cells, is normally absent, or expressed at very low levels in most somatic cells

 

. By
contrast, telomere maintenance is seen in virtually all types of cancers.

 

In 85% to 95% of
cancers, this is due to up-regulation of the enzyme telomerase.

 

A few tumors use other
mechanisms, termed alternative lengthening of telomeres, which probably depend on DNA
recombination.

 

Interestingly, in the progression from colonic adenoma to colonic
adenocarcinoma, early lesions had a high degree of genomic instability with low telomerase
expression, whereas malignant lesions had complex karyotypes with high levels of telomerase
activity, consistent with a model of telomere-driven tumorigenesis in human cancer. Several
other mechanisms of genomic instability are discussed later

244

Q image thumb

FIGURE 7-35 Sequence of events in the development of limitless replicative potential.
Replication of somatic cells, which do not express telomerase, leads to shortened telomeres.
In the presence of competent checkpoints, cells undergo arrest and enter nonreplicative
senescence. In the absence of checkpoints, DNA-repair pathways are inappropriately
activated, leading to the formation of dicentric chromosomes. At mitosis the dicentric
chromosomes are pulled apart, generating random double-stranded breaks, which then
activate DNA-repair pathways, leading to the random association of double-stranded ends
and the formation, again, of dicentric chromosomes. Cells undergo numerous rounds of this
bridge-fusion-breakage cycle, which generates massive chromosomal instability and
numerous mutations. If cells fail to re-express telomerase, they eventually undergo mitotic
catastrophe and death. Re-expression of telomerase allows the cells to escape the bridgefusion-
breakage cycle, thus promoting their survival and tumorigenesis.

245

Even with all the genetic abnormalities discussed above, solid tumors cannot enlarge beyond 1
to 2 mm in diameter unless they are____________. 

 vascularized

 

Like normal tissues, tumors require delivery
of oxygen and nutrients and removal of waste products; presumably the 1- to 2-mm zone
represents the maximal distance across which oxygen
, nutrients, and waste can diffuse from
blood vessels.

 

 

246

Cancer cells can stimulate neo-angiogenesis, during which new vessels sprout
from previously existing capillaries, or, in some cases, vasculogenesis, in which endothelial cells
are recruited from the bone marrow ( Chapter 3 ).

 

Tumor vasculature is abnormal, however.

 

T or F 
 

True

247

Describe the vacularization of tumors.

The vessels are leaky and dilated, and have a haphazard pattern of connection.
 

248

Neovascularization has a dual effect on tumor growth:

 

 

\

  • perfusion supplies needed nutrients and oxygen,
  • and newly formed endothelial cells stimulate the growth of adjacent tumor cells by secreting growth factors, such as insulin-like growth factors (IGFs), PDGF, and granulocytemacrophage

colony-stimulating factor. 

249

Angiogenesis is required not only for continued tumor
growth but also for access to the vasculature and hence for metastasis.

 

Angiogenesis is thus a
necessary biologic correlate of malignancy

 

T or F 

True

250

How do growing tumors develop a blood supply?

 

 

 

The emerging paradigm is that tumor
angiogenesis is controlled by the balance between angiogenesis promoters and inhibitors.
 

 

Early in their growth, most human tumors do not induce angiogenesis.

They remain small or in
situ, possibly for years, until the angiogenic switch terminates this stage of vascular quiescence. [105]

 

The molecular basis of the angiogenic switch involves increased production
of angiogenic factors and/or loss of angiogenic inhibitors.

 

251

The molecular basis of the angiogenic switch involves increased production
of angiogenic factors and/or loss of angiogenic inhibitors. 

These factors may be produced
directly by the tumor cells themselves or by inflammatory cells (e.g., macrophages) or other
stromal cells associated with the tumors
.

252

 Proteases, either elaborated by the tumor cells directly or from stromal cells in response to the tumor, are also involved in regulating the balance
between angiogenic and anti-angiogenic factor
s.

How?

Many proteases can release the
proangiogenic basic fibroblast growth factors (bFGF) stored in the ECM; conversely, three
potent angiogenesis inhibitors—angiostatin, endostatin, and vasculostatin
—are produced by
proteolytic cleavage of plasminogen, collagen, and transthyretin, respectively.

 

The angiogenic
switch is controlled by several physiologic stimuli, such as hypoxia.

Relative lack of oxygen
stimulates HIF1α, an oxygen-sensitive transcription factor mentioned above, which then
activates transcription of a variety of pro-angiogenic cytokines, such as VEGF and bFGF.

These factors create an angiogenic gradient that stimulates the proliferation of endothelial cells
and guides the growth of new vessels toward the tumor.

 

 

253

What is the role of Notch signaling pathway?

VEGF also increases the expression of
ligands that activate the Notch signaling pathway, which plays a crucial role in regulating the
branching and density of the new vessels ( Chapter 3 ). 

Both pro- and anti-angiogenic factors
are regulated by many other genes frequently mutated in cancer. For example, in normal cells,

254

For example in normal cell, p53 can stimulate expression of anti-angiogenic molecules such as thrombospondin-1, and
repress expression of pro-angiogenic molecules such as VEGF.

 

Thus, loss of p53 in tumor cells
not only removes the cell cycle checkpoints listed above but also provides a more permissive
environment for angiogenesis.
 

 

255

The transcription of VEGF is also influenced by signals from the
RAS-MAP kinase pathway, and mutations of RAS or MYC up-regulate the production of VEGF.
The mechanisms whereby bFGF, VEGF, and the Notch pathway work together to coordinate
angiogenesis were discussed in Chapter 3 . bFGF and VEGF are commonly expressed in a
wide variety of tumor cells, and elevated levels can be detected in the serum and urine of a
significant fraction of cancer patient
s.

 

Indeed, an anti-VEGF monoclonal antibody,
bevacizumab, has recently been approved for use in the treatment of multiple cancers. [106]
Another emerging strategy involves the use of antibodies that inhibit Notch activation. These
antibodies cause new vessels to be so malformed that they cannot deliver blood to the tumor
effectively.

256

What are the  biologic hallmarks of malignant tumors?

Invasion and metastasis are biologic hallmarks of malignant tumors.

They are the major cause
of cancer-related morbidity and mortality and hence are the subjects of intense scrutiny.
Studies in mice and humans reveal that although millions of cells are released into the
circulation each day from a primary tumor, only a few metastases are produced.

 

Indeed, tumor
cells can be frequently detected in the blood and marrow of patients with breast cancer who
have not, and do not ever, develop gross metastatic disease. 

257

Why is the metastatic process so
inefficient?

 Each step in the process is subject to a multitude of controls; hence, at any point in
the sequence the breakaway cell may not survive. [109]

For tumor cells to break loose from a
primary mass, enter blood vessels or lymphatics, and produce a secondary growth at a distant
site, they must go through a series of steps (summarized in Fig. 7-36 ).

 

 

258

For the purpose of this
discussion, the metastatic cascade will be divided into two phases:

 

  • (1) invasion of the extracellular matrix (ECM);
  • (2) vascular dissemination, homing of tumor cells, and colonization.

 

Subsequently, the molecular genetics of the metastatic cascade, as currently understood, will be presented

259

Q image thumb

FIGURE 7-36 The metastatic cascade. Sequential steps involved in the hematogenous
spread of a tumor.

260

The structural organization and function of normal tissues is to a great extent determined by
interactions between cells and the ECM. [110]

As we discussed in Chapter 3 , tissues are
organized into compartments separated from each other by two types of ECM:____________

 basement
membrane and interstitial connective tissue. 

 

Though organized differently, each of these
components of ECM is made up of collagens, glycoproteins, and proteoglycans. As shown in
Figure 7-36 , tumor cells must interact with the ECM at several stages in the metastatic
cascade.

 

 

261

 tumor cells must interact with the ECM at several stages in the metastatic
cascade.

 

A carcinoma must first :

  • breach the underlying basement membrane,
  • then traverse the interstitial connective tissue, and
  • ultimately gain access to the circulation by penetrating the vascular basement membrane.

 

This process is repeated in reverse when tumor cell emboli extravasate at a distant site.

 

262

Invasion of the ECM initiates the metastatic cascade and is an active process that can be resolved into several steps ( Fig. 7-37 ):

• Changes (“loosening up”) of tumor cell-cell interactions
• Degradation of ECM
• Attachment to novel ECM components
• Migration of tumor cells

263

Q image thumb

FIGURE 7-37 A–D, Sequence of events in the invasion of epithelial basement membranes
by tumor cells. Tumor cells detach from each other because of reduced adhesiveness, then
secrete proteolytic enzymes, degrading the basement membrane. Binding to proteolytically
generated binding sites and tumor cell migration follow.

264

Dissociation of cells from one another is often the result of alterations in___________

 

 intercellular adhesion
molecules. 

 

Normal cells are neatly glued to each other and their surroundings by a variety of
adhesion molecules. [111]

 

265

What mediates Cell-cell interactions are mediated by ?

Cell-cell interactions are mediated by the cadherin family of transmembrane glycoproteins.

E-cadherins mediate homotypic adhesions in epithelial tissue, thus serving to keep the epithelial cells together and to relay signals between the cells;
intracellularly the E-cadherins are connected to β-catenin and the actin cytoskeleton.

 

In several
epithelial tumors, including adenocarcinomas of the colon and breast, there is a downregulation
of E-cadherin expression.

 

Presumably, this down-regulation reduces the ability of
cells to adhere to each other and facilitates their detachment from the primary tumor and their
advance into the surrounding tissues. E-cadherins are linked to the cytoskeleton by the catenins, proteins that lie under the plasma membrane (see Fig. 7-33 ). 

266

The normal function of
E-cadherin is dependent on its linkage to _______

 

 

catenins.

 

In some tumors E-cadherin is normal, but its
expression is reduced because of mutations in the gene for α catenin

267

The second step in invasion is ________________. 

local degradation of the basement membrane and interstitial
connective tissue

268

How do tumor cells invace locally the basement membrane?

Tumor cells may either secrete proteolytic enzymes themselves or induce stromal cells (e.g., fibroblasts and inflammatory cells) to elaborate proteases.

 

269

Many different
families of proteases, such as____________

have been implicated in tumor cell invasion. 

  •  matrix metalloproteinases (MMPs),
  • cathepsin D,
  • and urokinase plasminogen activator

270

How do MMPs regulate tumor invasion?

MMPs regulate tumor
invasion not only by remodeling insoluble components of the basement membrane and
interstitial matrix but also by releasing ECM-sequestered growth factors.

 

Indeed, cleavage
products of collagen and proteoglycans also have chemotactic, angiogenic, and growthpromoting
effects. [112]

 

For example, MMP9 is a gelatinase that cleaves type IV collagen of the epithelial and vascular basement membrane and also stimulates release of VEGF from ECMsequestered pools.

 

Benign tumors of the breast, colon, and stomach show little type IV collagenase activity, whereas their malignant counterparts overexpress this enzyme.

 

Concurrently, the concentrations of metalloproteinase inhibitors are reduced so that the
balance is tilted greatly toward tissue degradation. Indeed, overexpression of MMPs and other
proteases has been reported for many tumors.

 

 

271

However, recent in vivo imaging experiments
have shown that tumor cells can adopt a second mode of invasion, termed what?

 ameboid migration. [113]

 

In this type of migration the cell squeezes through spaces in the matrix instead
of cutting its way through it.

This ameboid migration is much quicker, and tumor cells seem to be able to use collagen fibers as high-speed railways in their travels.

 

Tumor cells, in vitro at least,
seem to be able to switch between the two forms of migration, perhaps explaining the
disappointing performance of MMP inhibitors in clinical trials.

272

The third step in invasion involves changes in _____________
 

attachment of tumor cells to ECM proteins .

 

Normal epithelial cells have receptors, such as integrins, for basement membrane laminin and
collagens that are polarized at their basal surface;
these receptors help to maintain the cells in
a resting, differentiated state.

 

Loss of adhesion in normal cells leads to induction of apoptosis, while, not surprisingly, tumor cells are resistant to this form of cell death.

 

Additionally, the matrix
itself is modified in ways that promote invasion and metastasis.

For example, cleavage of the
basement membrane proteins collagen IV and laminin by MMP2 or MMP9 generates novel sites
that bind to receptors on tumor cells and stimulate migration.

273

What is the final step of invasion?

Locomotion is the final step of invasion, propelling tumor cells through the degraded basement
membranes and zones of matrix proteolysis.

 

Migration is a complex, multistep process that
involves many families of receptors and signaling proteins that eventually impinge on the actin
cytoskeleton.

 

Cells must attach to the matrix at the leading edge, detach from the matrix at the
trailing edge, and contract the actin cytoskeleton to ratchet forward.

Such movement seems to
be potentiated and directed by tumor cell–derived cytokines, such as autocrine motility factors.


In addition, cleavage products of matrix components (e.g., collagen, laminin) and some growth
factors (e.g., IGFs I and II) have chemotactic activity for tumor cells.

 

Furthermore, proteolytic
cleavage liberates growth factors bound to matrix molecules.

 

Stromal cells also produce
paracrine effectors of cell motility, such as hepatocyte growth factorscatter factor, which bind
to receptors on tumor cells.

Concentrations of hepatocyte growth factor–scatter factor are elevated at the advancing edges of the highly invasive brain tumor glioblastoma multiforme,
supporting their role in motility.

274

It has become clear in recent years that the ECM and stromal cells surrounding tumor cells do
not merely represent a static barrier for tumor cells to traverse but instead represent a varied
environment in which reciprocal signaling between tumor cells and stromal cells may either
promote or prevent tumorigenesis and/or tumor progression
. [24] 

 

T or F

True

 

Stromal cells that interact with
tumors include innate and adaptive immune cells (discussed later), as well as fibroblasts.

A
variety of studies have demonstrated that tumor-associated fibroblasts exhibit altered
expression of genes that encode ECM molecules, proteases, protease inhibitors, and various
growth factors.

Thus, tumor cells live in a complex and ever-changing milieu composed of ECM,
growth factors, fibroblasts, and immune cells, with significant cross-talk among all the
components. The most successful tumors may be those that can co-opt and adapt this
environment to their own nefarious ends.

275

Once in the circulation, tumor cells are vulnerable to destruction by a variety of mechanisms,
including mechanical shear stress, apoptosis stimulated by loss of adhesion, (which has been
termed anoikis), and innate and adaptive immune defenses. The details of tumor immunity are
considered later.

276

Within the circulation, tumor cells tend to aggregate in clumps.

This is favored by homotypic
adhesions among tumor cells as well as heterotypic adhesion between tumor cells and blood
cells, particularly platelets
(see Fig. 7-36 ).

 

Formation of platelet-tumor aggregates may
enhance tumor cell survival and implantability.

 

Tumor cells may also bind and activate
coagulation factors, resulting in the formation of emboli.

 

Arrest and extravasation of tumor
emboli at distant sites involves adhesion to the endothelium, followed by egress through the
basement membrane.

 

Involved in these processes are adhesion molecules (integrins, laminin
receptors) and proteolytic enzymes, discussed earlier. Of particular interest is the CD44 adhesion molecule, which is expressed on normal T lymphocytes and is used by these cells to migrate to selective sites in the lymphoid tissue.

Such migration is accomplished by the binding
of CD44 to hyaluronate on high endothelial venules, and overexpression of CD44 may favor
metastatic spread.

At the new site, tumor cells must proliferate, develop a vascular supply, and
evade the host defenses

277

The site at which circulating tumor cells leave the capillaries to form secondary deposits is
related, in part, to the anatomic location of the primary tumor, with most metastases occurring in
the______________-

 

 first capillary bed available to the tumor.

 

 

 

278

Many observations, however, suggest that natural
pathways of drainage do not wholly explain the distribution of metastases

. For example,
prostatic carcinoma preferentially spreads to bone, bronchogenic carcinomas tend to involve
the adrenals and the brain, and neuroblastomas spread to the liver and bones.

Such organ
tropism may be related to the following mechanisms:

  • • Because the first step in extravasation is adhesion to the endothelium, tumor cells may have adhesion molecules whose ligands are expressed preferentially on the endothelial cells of the target organ. Indeed, it has been shown that the endothelial cells of the vascular beds of various tissues differ in their expression of ligands for adhesion molecules.
  • • Chemokines have an important role in determining the target tissues for metastasis. For instance, some breast cancer cells express the chemokine receptors CXCR4 and CCR7. [114] The chemokines that bind to these receptors are highly expressed in tissues to which breast cancers commonly metastasize. Blockage of the interaction between CXCR4 and its receptor decreases breast cancer metastasis to lymph nodes and lungs. Some target organs may liberate chemoattractants that recruit tumor cells to the site. Examples include IGFs I and II.

 

  • • In some cases, the target tissue may be a nonpermissive environment—unfavorable soil, so to speak, for the growth of tumor seedlings. For example, though well vascularized, skeletal muscles are rarely the site of metastases.

279

Despite their “cleverness” in escaping their sites of origin, tumor cells are quite inefficient in
colonizing distant organs.

Millions of tumors cells are shed daily from even small tumors.

These
cells can be detected in the bloodstream and in small foci in the bone marrow, even in patients
that never develop gross metastatic lesions.

Indeed, the concept of dormancy, referring to the
prolonged survival of micrometastases without progression, is well described in melanoma and
in breast and prostate cancer.

Although the molecular mechanisms of colonization are just beginning to be unraveled in mouse models, a constant pattern seems to be that tumor cells
secrete cytokines, growth factors, and ECM molecules that act on the resident stromal cells,

which in turn make the metastatic site habitable for the cancer cell. [115]

 

Give an example.

 

 

For example, breast
cancer metastases to bone are osteolytic because of the activation of osteoclasts in the metastatic site.

Breast cancer cells secrete parathyroid hormone–related protein (PTHRP), which stimulates osteoblasts to make RANK ligand (RANKL).

RANKL then activates osteoclasts, which degrade the bone matrix and release growth factors embedded within it, like IGF and TGF-β. With a better molecular understanding of the mechanisms of metastasis our ability to target them therapeutically will be greatly enhanced

280

Why do only some tumors metastasize? What are the genetic changes that allow metastases?
Why is the metastatic process so inefficient?

Several competing theories have been proposed
to explain how the metastatic phenotype arises.

 

The clonal evolution model suggest that, as
mutations accumulate in genetically unstable cancer cells and the tumor become
heterogeneous ( Fig. 7-38A ), a subset of tumor cell subclones develop the right combination of
gene products to complete all the steps involved in metastasis. Thus, metastatic subclones
result from clonal evolution, and it is only the rare cell that acquires all the necessary genetic
alterations and can complete all the steps.

 

However, recent experiments, in which gene
expression profiles of primary tumors and metastatic deposits have been compared, challenge
this hypothesis.

 

For example, a subset of breast cancers has a gene expression signature
similar to that found in metastases, although no clinical evidence for metastasis is apparent. In
these tumors it seems that most if not all cells develop a predilection for metastatic spread
during early stages of carcinogenesis. Metastases, according to this view, are not dependent
on the stochastic generation of metastatic subclones postulated above.

 

The alternative
hypothesis suggested by these data is that metastasis is the result of multiple abnormalities that
occur in many, perhaps most, cells of a primary tumor, and perhaps early in the development of
the tumor ( Fig. 7-38B and C ). Such abnormalities give most cells within the tumor a general
predisposition for metastasis, often called the “metastasis signature.” [116]

This signature may
involve not only properties intrinsic to the cancer cells but also the characteristics of their
microenvironment, such as the components of the stroma, the presence of infiltrating immune
cells, and angiogenesis ( Fig. 7-38D ). It should be noted, however, that gene expression
analyses like those described above would not detect a small subset of metastatic subclones
within a large tumor. Perhaps both mechanisms are operative, with aggressive tumors acquiring
a metastases-permissive gene expression pattern early in tumorigenesis that requires some
additional random mutations to complete the metastatic phenotype.

 

A third hypothesis suggests
that background genetic variation, and the resulting variation in gene expression
, in the human
population contributes to the generation of metastases. In mouse models, cancers induced with
the same oncogenic mutations can have very different metastatic outcomes depending on the strain (i.e., background genetics) of the mouse used. Even very strong oncogenes can be
significantly affected by background genetics. The fourth hypothesis is a corollary of the tumor
stem cell hypothesis, which suggests that if tumors derive from rare tumor stem cells,
metastases require the spread of the tumor stem cells themselves

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Q image thumb

FIGURE 7-38 Mechanisms of metastasis development within a primary tumor. A
nonmetastatic primary tumor is shown (light blue) on the left side of all diagrams. Four
models are presented: A, Metastasis is caused by rare variant clones that develop in the
primary tumor; B, Metastasis is caused by the gene expression pattern of most cells of the
primary tumor, referred to as a metastatic signature; C, A combination of A and B, in which
metastatic variants appear in a tumor with a metastatic gene signature; D, Metastasis
development is greatly influenced by the tumor stroma, which may regulate angiogenesis,
local invasiveness, and resistance to immune elimination, allowing cells of the primary tumor,
as in C, to become metastatic.

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One open question in the field is, are there genes whose principal or sole contribution to
tumorigenesis is to control metastasis? This question is of more than academic interest,
because if altered forms of certain genes promote or suppress the metastatic phenotype, their
detection in a primary tumor would have both prognostic and therapeutic implications.

 

Since
metastasis is a complex phenomenon involving a variety of steps and pathways described
above, it is thought that, unlike transformation, in which a subset of proteins like p53 and RB
seem to play a key role, genes that function as “metastasis oncogenes” or “metastatic
suppressors” are rare.

 

 

What is metastasis oncogenes?

A metastasis suppressor gene is defined as a gene whose loss promotes the development of metastasis without an effect on the primary tumor.

Accordingly,
expression of a metastasis oncogene favors the development of metastasis without effect upon
the primary tumo
r.

At least a dozen genes lost in metastatic lesions have been confirmed to
function as “metastasis suppressors”. [117,] [118]

 

Their molecular functions are varied and not
yet completely clear; however, most appear to affect various signaling pathways. Interestingly,
recent work has suggested that two miRNAs, mir335 and mir126, suppress the metastasis of
breast cancer, while a second set (mir10b) promotes metastasis.

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Among candidates for metastasis oncogenes are_____________ which encode transcription
factors whose primary function is to promote a process called epithelial-tomesenchymal
transition (EMT).
[88] In EMT, carcinoma cells down-regulate certain epithelial markers (e.g., cadherin) and up-regulate certain mesenchymal markers (e.g., vimentin and smooth muscle
actin). These changes are believed to favor the development of a promigratory phenotype that
is essential for metastasis. Loss of E-cadherin expression seems to be a key event in EMT, and
SNAIL and TWIST are transcriptional repressors that down-regulate E-cadherin
expression. [121] EMT has been documented mainly in breast cancers; whether this is a
general phenomenon remains to be established.

SNAIL and TWIST,

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