INSENSITIVITY TO

GROWTH
INHIBITORY BY-

SIGNALS DR. ISHITA SINGHAL

1. INTRODUCTION
2. ACTIVATORS AND INHIBITORS
3. ALTERATIONS IN CELL CYCLE CONTROL PROTEINS IN CANCER CELLS
4. INSENSITIVITY TO GROWTH INHIBITORY SIGNALS
5. RB GENE: Governor Of The Cell Cycle
6. TP53 GENE: Guardian Of The Genome
7. TRANSFORMING GROWTH FACTOR-B PATHWAY
8. APC-B-CATENIN PATHWAYS
9. REFERENCES

INDEX
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INTRODUCTI
ON

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 Cell proliferation is fundamental to:

1. Development.
2. Maintenance of steady-state tissue homeostasis.
3. Replacement of dead or damaged cells.

 The key elements of cellular proliferation to daughter cells through

mitosis and cytokinesis are:

1. Accurate DNA replication.

2. Coordinated synthesis of all other cellular constituents.
3. Equal apportionment of DNA and other cellular organelles.

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 The sequence of events that results in cell division is called the cell cycle.

 It consists of:

1. G1 (Presynthetic growth) Phase

2. S (DNA synthesis) Phase
3. G2 (Premitotic growth) Phase
4. M (Mitotic) Phase

 Quiescent cells that are not actively cycling are said to be in the G0 state.

 Cells can enter G1 either from the G0 quiescent cell pool, or after
completing a round of mitosis, as for continuously replicating cells.

 Non-fidelity of DNA replication, or cofactor deficiency result in arrest at

the various transition points.
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ACTIVATORS
AND
INHIBITORS

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 Cell cycle progression is driven by:

1. Proteins called cyclins named for the cyclic nature of their

production and degradation.
2. Cyclin-associated enzymes called cyclin dependent kinases (CDKs).

 CDKs acquire the ability to phosphorylate protein substrates by forming

complexes with the relevant cyclins.

 Transiently increased synthesis of a particular cyclin leads to increased

kinase activity of the appropriate CDK binding partner.

 As the CDK completes its round of phosphorylation, the associated cyclin

is degraded and the CDK activity abates.

 Thus, as cyclin levels rise and fall, the activity of associated CDKs
likewise wax and wane. FIRST UP
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 Embedded in the cell cycle are surveillance mechanisms primed to sense
DNA or chromosomal damage.

 These quality control checkpoints ensure that cells with genetic

imperfections do not complete replication:

1. The G1-S checkpoint monitors the integrity of DNA before

irreversibly committing cellular resources to DNA replication.
2. The G2-M restriction point ensures that there has been accurate
genetic replication before the cell actually divides.

 When cells do detect DNA irregularities, checkpoint activation delays

cell cycle progression and triggers DNA repair mechanisms.

 If the genetic derangement is too severe to be repaired, the cells will

undergo apoptosis; alternatively, they may enter a non replicative state
called senescence. FIRST UP
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 Enforcing the cell cycle checkpoints is the job of CDK
inhibitors(CDKIs).

 There are several different CDKIs:

1. The Cip/Kip (CDK interacting protein/Kinase inhibitory protein)

family broadly inhibits multiple CDKs and includes:
a) p21 (CDKN1A)
b) p27 (CDKN1B)
c) p57 (CDKN1C)

2. The INK4a/ARF (Inhibitor of Kinase 4/Alternative Reading Frame)

family has selective effects on cyclin CDK4 and cyclin CDK6 and
includes:
a) p15 (CDKN2B)
b) p16 (CDKN2A)
c) p18 (CDKN2C) FIRST UP
d) p19 (CDKN2D) CONSULTANTS
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 While cyclins arouse the CDKs, CDK inhibitors (CDKIs), of which
there are many, silence the CDKs and exert negative control over the
cell cycle.

 Defective CDKI checkpoint proteins allow cells with damaged DNA to

divide, resulting in mutated daughter cells with the potential of
developing into malignant tumors.

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 ALTERATIONS
IN CELL CYCLE
CONTROL
PROTEINS IN
CANCER
CELLS
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 The major cancer-associated mutations that affect the G1/S
checkpoint can be broadly grouped into two classes:

1. Gain-of-function mutations involving CDK4 and D cyclin:

a) Mishaps increasing the expression of cyclin D or CDK4 are

common events in neoplastic transformation.
b) The cyclin D genes are overexpressed in many cancers.

2. Loss-of-function mutations involving CDKIs:

a) CDKIs that inhibit cyclin D/CDK complexes are frequently

mutated or otherwise silenced in many human malignancies.

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INSENSITIVITY
TO GROWTH
INHIBITORY
SIGNALS

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 In principle, anti-growth signals can prevent cell proliferation by several
complementary mechanisms.

 The signal may cause dividing cells to enter G0, where they remain until
external cues prod their re-entry into the proliferative pool.

 Alternatively, the cells may enter a postmitotic, differentiated pool and

lose replicative potential.

 Non-replicative senescence, is another mechanism of escape from

sustained cell growth.

 And, as a last-ditch effort, the cells may be programmed for death by

apoptosis.

 Therefore, tumor suppressor genes have all these “tricks” in their

toolbox designed to halt wayward cells from becoming malignant. FIRST UP
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RB GENE:
GOVERNOR
OF THE
CELL CYCLE

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 RB, a key negative regulator of the cell cycle, is directly or indirectly
inactivated in most human cancers.

 The retinoblastoma gene (RB) was the first tumor suppressor gene to be
discovered.

 The discovery of tumor suppressor genes was accomplished by the study

of a rare disease—in this case, retinoblastoma, an uncommon
childhood tumor.

 The tumor is being transmitted as an autosomal dominant trait.

 It is of two types:

1. 60% sporadic
2. 40% familial
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 To account for the sporadic and familial occurrence of an identical
tumor, Knudson, in 1974, proposed his now famous two-hit
hypothesis, which in molecular terms can be stated as follows:

1. Two mutations (hits) are required to produce retinoblastoma.

2. These involve the RB gene, which has been mapped to

chromosomal locus 13q14.

3. Both of the normal alleles of the RB locus must be inactivated for

the development of retinoblastoma.

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Alfred George Knudson, Jr. 

(August 9, 1922 – July 10, 2016)

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 In Sporadic Cases:

1. Both normal RB alleles are lost by somatic mutation in one of the

retinoblasts.
2. In the end a retinal cell that has lost both of the normal copies of the
RB gene becomes cancerous.

 In Familial Cases:

1. Children inherit one defective copy of the RB gene in the germ line;
the other copy is normal.
2. Retinoblastoma develops when the normal RB gene is lost in
retinoblasts as a result of somatic mutation.
3. Because in retinoblastoma families a single germ line mutation is
sufficient to transmit disease risk, the trait has an autosomal
dominant inheritance pattern.
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 Although the loss of normal RB genes initially was discovered in
retinoblastomas, it is now evident that biallelic loss of this gene is a
fairly common feature of several tumors like:

1. Breast cancer
2. Small cell cancer of the lung
3. Bladder cancer

 Patients with familial retinoblastoma also are at greatly increased

risk for developing:

1. Osteosarcomas
2. Some soft-tissue sarcomas.

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 SMALL CELL CANCER
BREAST CANCER BLADDER CANCER
OF THE LUNG

SOFT TISSUE
OSTEOSARCOMA
SARCOMA
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 The function of the RB protein is to regulate the G1/S checkpoint, the
portal through which cells must pass before DNA replication
commences.

 In the G1 phase, diverse signals are integrated to determine whether the

cell should progress through the cell cycle, or exit the cell cycle and
differentiate.

 RB, is a DNA-binding protein that serves as a point of integration for

these diverse signals, which ultimately act by altering the
phosphorylation state of RB.

 Specifically, signals that promote cell cycle progression lead to the

phosphorylation and inactivation of RB, while those that block cell cycle
progression act by maintaining RB in an active hypophosphorylated
state.
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 Mechanism:

1. The initiation of DNA replication requires the activity of cyclin

E/CDK2 complexes, and expression of cyclin E is dependent on the
E2F(Elongation Factor 2) family of transcription factors.
2. 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.
3. Hypophosphorylated RB blocks E2F-mediated transcription in 2
ways:

a) It sequesters E2F, preventing it from interacting with other

transcriptional activators.
b) RB recruits chromatin remodeling proteins to make DNA
insensitive to transcription factors, such as histone deacetylases
and histone methyltransferases, which bind to the promoters of
E2F-responsive genes such as cyclin E. FIRST UP
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 This situation is changed on mitogenic signaling:

1. Growth factor signaling leads to cyclin D expression and activation

of cyclin D–CDK4/6 complexes.

2. These complexes phosphorylate RB, inactivating the protein and

releasing E2F to induce target genes such as cyclin E.

3. Cyclin E/CDK complexes then stimulate DNA replication and

progression through the cell cycle.

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

5. During the ensuing M phase, the phosphate groups are removed

from RB by cellular phosphatases, regenerating the
hypophosphorylated form of RB. FIRST UP
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Q: Why RB is not mutated in every cancer?

Mutations in other genes that control RB phosphorylation can mimic the

effect of RB loss and are commonly found in many cancers that have
normal RB genes.

For example:

1. Mutational activation of CDK4 and overexpression of cyclin D favor

cell proliferation by facilitating RB phosphorylation and inactivation.
Indeed, cyclin D is overexpressed in many tumors because of
amplification or translocation of the cyclin D1 gene.
2. Mutational inactivation of genes encoding CDKIs also can drive the
cell cycle by removing important brakes on cyclin/ CDK activity.
3. CDKN2A gene, which encodes the CDK inhibitor p16, is an extremely
common target of deletion or mutational inactivation in human tumors.
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 The loss of normal cell cycle control is central to malignant
transformation and that at least one of the four key regulators of the cell
cycle (p16, cyclin D, CDK4, RB) is mutated in most human cancers.

 Notably, in cancers caused by certain oncogenic viruses, this is achieved

through direct targeting of RB by viral proteins.

For example:

1. The human papillomavirus (HPV) E7 protein binds to the

hypophosphorylated form of RB, preventing it from inhibiting the
E2F transcription factors.

2. Thus, RB is functionally deleted, leading to uncontrolled growth.

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Thomas S et al (2015) stated that the overexpression of Rb protein noted
in oral cancer, with an increase in well and moderately differentiated
tumors suggest a possible role of Rb in differentiation. The high expression
of Rb in patients with combined habits of Paan chewing, smoking and
alcohol consumption indicates that Rb pathway may be altered in habit-
related oral malignancies.

Thomas S, Balan A, Balaram P. The expression of retinoblastoma tumor suppressor protein in oral cancers and precancers: A FIRST UP
clinicopathological study. Dent Res J. 2015;12(4):307–14. CONSULTANTS
 TP53:
GUARDIAN OF
THE GENOME

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 The p53 protein is a transcription factor that thwarts neoplastic
transformation by three interlocking mechanisms:

1. Activation of temporary cell cycle arrest (termed quiescence)

2. Induction of permanent cell cycle arrest (termed senescence)
3. Triggering of programmed cell death (termed apoptosis)

 p53 can be viewed as a central monitor of internal stress, directing the

stressed cells toward one of these pathways.

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 A variety of stresses trigger the p53 response pathways:

1. Anoxia
2. Inappropriate oncoprotein activity
3. DNA damage

 By managing the DNA damage response, p53 plays a central role in

maintaining the integrity of the genome.

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 In healthy cells, p53 has a short half-life (20 minutes) because of its
association with MDM2(Mouse double minute 2 homolog), a protein that
targets p53 for destruction.

 When the cell is stressed, by an assault on its DNA, “sensors” that

include protein kinases such as ATM (Ataxia Telangiectasia Mutated)
are activated.

 These activated sensors catalyse post translational modifications in p53

that release it from MDM2, increasing its half-life and enhancing its
ability to drive the transcription of target genes.

 Hundreds of genes whose transcription is triggered by p53 have been

found.

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 These genes suppress neoplastic transformation by three
mechanisms:

1. p53-mediated cell cycle arrest may be considered the primordial

response to DNA damage.
2. p53-induced senescence is a form of permanent cell cycle arrest.
3. p53-induced apoptosis of cells with irreversible DNA damage is the
ultimate protective mechanism against neoplastic transformation.

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1. p53-mediated cell cycle arrest may be considered the primordial
response to DNA damage.

a) It occurs late in the G1 phase and is caused mainly by p53-dependent

transcription of the CDKI gene p21.

b) The p21 protein inhibits cyclin–CDK complexes and prevents

phosphorylation of RB, thereby arresting cells in the G1 phase.

c) Such a pause in cell cycling is welcome, because it gives the cells

“breathing time” to repair DNA damage.

d) If DNA damage is repaired successfully, p53 up regulates

transcription of MDM2, leading to its own destruction and relief of
the cell cycle block.

e) If the damage cannot be repaired, the cell may enter p53- induced
senescence or undergo p53-directed apoptosis. FIRST UP
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2. p53-induced senescence is a form of permanent cell cycle arrest:

a) It is characterized by specific changes in morphology and gene

expression that differentiate it from quiescence or reversible cell
cycle arrest.

b) Senescence requires activation of p53 and/or Rb and expression of

their mediators, such as the CDKIs.

c) The mechanisms of senescence are unclear but seem to involve

global chromatin changes, which drastically and permanently alter
gene expression.

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

a) It is mediated by up regulation of several pro-apoptotic genes,

including BAX and PUMA.

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 Confirming the importance of TP53 in controlling carcinogenesis, more
than 70% of human cancers have a defect in this gene.

 Biallelic abnormalities of the TP53 gene are found in virtually every

type of cancer like:

1. Carcinomas of the lung

2. Colon
3. Breast

—the three leading causes of cancer deaths.

 In most cases, mutations affecting both TP53 alleles are acquired in

somatic cells.

 In other tumors, such as certain sarcomas, the TP53 gene is intact but
p53 function is lost because of amplification and overexpression of the
MDM2 gene, which encodes a potent inhibitor of p53. FIRST UP
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 As with RB, normal p53 also can be rendered non-functional by certain
oncogenic DNA viruses.

 Proteins encoded by oncogenic HPVs, hepatitis B virus, and possibly

Epstein-Barr virus can bind to normal p53 and nullify its protective
function.

 Thus, DNA viruses can subvert two of the best understood tumor
suppressors, RB and p53.

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de Oliveira MG (2012) stated that the retinoblastoma (Rb) and p53 genes play
a fundamental role in cell cycle mechanisms, and their deregulation is related to
many steps of oral cancer carcinogenesis. He evaluated the expression of the
p53 and Rb proteins in malignant and pre-malignant oral cavity lesions. The
samples were processed by immunohistochemistry. Seventy-one patients were
studied. Of the samples studied, 59.4% were oral squamous cell carcinoma
(OSCC) and 40.6% were pre-neoplastic lesions (leukoplakia and actinic
cheilitis). OSCC presented higher expression of Rb protein compared to pre-
malignant lesions. Pre-neoplastic lesions presented higher expression of p53
protein compared to OSCC lesions. Despite the small number of samples, the
expression of these cell cycle biomarkers (p53 and Rb protein) in excisional
biopsies suggests that molecular lesion assessment can determine pre-malignant
lesions, and that its use may improve the clinical and surgical treatment of early
lesions. Thus, p53 protein expression may be related to the early steps of
carcinogenesis in OSCC. Finally, a higher Rb expression was also observed in
malignant lesions.
de Oliveira MG, Ramalho LM, Gaiao L, Pozza DH, de Mello RA. Retinoblastoma and p53 protein expression in pre-malignant oral FIRST UP
lesions and oral squamous cell carcinoma. Mol Med Rep. 2012;6:163–6. CONSULTANTS
TRANSFORMI
NG GROWTH
FACTOR-B
PATHWAY

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 TGF-β, is a member of a family of dimeric growth factors that includes
bone morphogenetic proteins and activins.

 In most normal epithelial, endothelial, and hematopoietic cells, TGF-β is

a potent inhibitor of proliferation.

 It regulates cellular processes by binding to a complex composed of

TGF-β receptors I and II.

 Dimerization of the receptor upon ligand binding leads to a cascade of

events that result in the transcriptional activation of CDKIs with growth-
suppressing activity, as well as repression of growth-promoting genes
such as MYC, CDK2, CDK4, and those encoding cyclins A and E.

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 In many forms of cancer, the growth-inhibiting effects of the TGF-β
pathways are impaired by mutations affecting TGF-β signaling.

 These mutations may alter the type II TGF-β receptor that serve to
transduce antiproliferative signals from the receptor to the nucleus.

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

1. Colon
2. Stomach
3. Endometrium

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 The TGF-B cell signaling pathway plays a complex role in cancer
development, progression, and metastasis.

 SMADs function in cell signal transduction from TGFB1 ligands to

activate gene transcription.

 Binding of the TGFB1 ligand dimer to the TGF-beta receptor type-2

(TGFBR2) promotes dimerization of TGFBR2 with TGFBR1 and results
in transphosphorylation of TGFBR1.

 The activated TGFBR1 activates R-SMADs (SMAD2 and SMAD3) via

phosphorylation.

 SMAD2 and SMAD3 trimerize with SMAD4.

 The SMAD trimer enters the nucleus to activate gene transcription and
promote cell growth and survival. FIRST UP
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 APC-B-
CATENIN
PATHWAYS

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 The rare hereditary disease Adenomatous Polyposis Coli (APC) is
characterized by the development of numerous adenomatous polyps in
the colon that have a very high incidence of transformation into colonic
cancers.

 They consistently show loss of a tumor suppressor gene called APC,

which exerts antiproliferative effects in an unusual manner.

 It encodes a cytoplasmic protein whose dominant function is to regulate

the intracellular levels of β-catenin, a protein with many functions and
an important component of the so-called WNT signaling pathway that
regulates cell proliferation. The name WNT is a portmanteau created
from the name Wingless and the name Int-1.

 β-catenin binds to the cytoplasmic portion of E-cadherin and can also

translocate to the nucleus and activate cell proliferation. Here the focus
is on the latter function of this protein. FIRST UP
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 WNT is a soluble factor that can induce cellular proliferation.

 It does so by binding to its receptor and transmitting signals that prevent

the degradation of β-catenin, allowing it to translocate to the nucleus,
where it acts as a transcriptional activator in conjunction with another
molecule, called TCF(T-Cell Factor).

 In quiescent cells, which are not exposed to WNT, cytoplasmic β-catenin

is degraded by a destruction complex, of which APC is an integral part.

 With loss of APC in malignant cells, β-catenin degradation is prevented,

and the WNT signaling response is inappropriately activated in the
absence of WNT.

 This leads to transcription of growth-promoting genes, such as cyclin D1

and MYC, as well as transcriptional regulators that repress E-cadherin
expression and thus reduce contact inhibition. FIRST UP
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 APC behaves as a typical tumor suppressor gene.

 Persons born with one mutant allele typically are found to have hundreds
to thousands of adenomatous polyps in the colon by their teens or 20s;
these polyps show loss of the other APC allele.

 Almost invariably, one or more polyps undergo malignant

transformation.

 APC mutations are seen in 70% to 80% of sporadic colon cancers.

 Colonic cancers that have normal APC genes show activating mutations
of β-catenin that render them refractory to the degrading action of APC.

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REFERENCES

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1. ROBBINS BASIC PATHOLOGY 9TH EDITION

2. Thomas S, Balan A, Balaram P. The expression of

retinoblastoma tumor suppressor protein in oral cancers and
precancers: A clinicopathological study. Dent Res J.
2015;12(4):307–14.

3. de Oliveira MG, Ramalho LM, Gaiao L, Pozza DH, de Mello

RA. Retinoblastoma and p53 protein expression in pre-
malignant oral lesions and oral squamous cell carcinoma. Mol
Med Rep. 2012;6:163–6.

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THANK YOU