o (C712R)

o (C706F) ∆703-37

o (R661W) ∆738-75

∆N480
∆127-66 ∆829-88

1 A B 928

N-terminal Small pocket C-terminal

Figure 1: The common mutation sites in the retinoblastoma protein (pRb). pRb is protein made up
of 928 amino acids and three structural regions (N-terminal, small pocket and C-terminal). Mutant
pRb protein often arises due to deletion (illustrated by ∆) and nonsense mutations (illustrated by o).
Most mutations are clustered at the B small pocket domain. Adapted from (Dick, 2007).
 CELL CYCLE
mtpRb PROGRESSION

Cyclin A DNA REPLICATION

Cyclin E AND NUCLEOTIDE
E2F Cdk1 SYNTHESIS
E2F1
PROMOTER TARGET GENES DNA
E2F2
polymerase 1
E2F3
Thymidine
Skp2
kinase
HP1
Thymidylate
PROTO- synthase
ONCOGENES Dihydrofolate
reductase
b-myb
c-myb
c-myc
c-myb

Figure 2: Regulation of E2F-dependent gene transcription by retinoblastoma protein (pRb). Mutant

pRb (mtpRb) cannot repress E2F mediated gene expression. Thus, overexpression of E2F target
genes which are involved in cell cycle progression, proto-oncogenes activation and DNA replication
as well as nucleotide synthesis aid in tumorigenesis. Adapted from (Sachdeva and O'Brien, 2012).
 CELL ADHESION CELL CYCLE CONTROL CHROMOSOMAL INSTABILITY

mtpRb mtpRb mtpRb

↓ E-cadherin ↑ E2F
↑ Mad2
Merlin detachment
↑ MMP ↑ CAP-D3
Integrin switch ↑ Cyclin A
↑ Cyclin E
↑ Cdk1 DNA damage
EMT
↑ Skp2 Mitotic error
Aneuploidy

Metastasis
G1/S progression

Figure 3: The various cellular processes affected by mutant pRb (mtpRb). mtpRb decreases cell
adhesion. mtpRb decreases the expression of E-cadherin, stimulates the detachment of Merlin
cytoskeletal adaptor from the plasma membrane, triggers integrin switch from integrin α10 to
integrin β4 and elevates metalloproteinases (MMP) synthesis that remodels the extracellular matrix.
All these changes trigger destabilization of adherens junction causing epithelial-mesenchymal
transition (EMT) of cancer cell; thus, metastasis. mtpRb causes deregulation in the cell cycle control.
mtpRb cannot repress E2F transcription factor leading to stimulation of multiple genes, resulting
G1/S progression and over proliferation. mtpRb also cause chromosomal instability. Rise in the
concentration of Mad2 due to overexpression of E2F triggers aneuploidy and DNA breaks. Elevation
in condensin II (CAP-D3) binding to chromosome due to mtpRb impairs chromosome condensation;
hence resulting in DNA damage and mitotic error.
 R248 R249

G245 R273

R175 R282

1 DNA BINDING DOMAIN 393

N-terminal Central core C-terminal

Figure 4: The structure of p53 protein. p53 is protein made up of 393 amino acids and three main
regions (N-terminal, central core and C-terminal). Mutant p53 protein often arises due to missense
mutations involving 6 hotspots (R175, G245, R248, R249, R273 and R282) which are concentrated at
the DNA-binding domain located at the central core region. Adapted from (Zhang et al, 2016).
 CELL CYCLE CONTROL APOPTOSIS GENOME INSTABILITY

mtp53

mtp53 ↓ Bax ↓ Fas ↓ IGFBP3

↓ PUMA
↓ p21Cip1 ↓ 14-3-3σ
mtp53
↓ NOXA
Cas 8
↓ p53AIP1 ↑ IGF1&2 ↓ Tap63
↓ p53R2
↑ cyclinE/Cdk2 ↑ Cdc25C
Cas 3&7
↓ cyt c
↓ RR ↓ BubR1
↑ phos-pRb cyclinB/Cdk1

Cas 9 ↓ DNA repair Aneuploidy

↑ G1/S ↑ G2/M

Cas 3

↓ apoptosis

Figure 5: The various cellular processes affected by mutant p53 (mtp53). mtp53 affects cell cycle
control resulting in over proliferation. mtp53 decreases p21 Cip1 expression leading to activation of
cyclinE/Cdk2. Activated cyclinE/Cdk2 triggers hyperphosphorylation of pRb (phos-pRb) resulting in
G1/S progression. mtp53 decreases 14-3-3σ synthesis causing elevation of Cdc25C phosphatase level
in the nucleus. Cdc25C can trigger the dephosphorylation and activation of cyclinB/Cdk1 that hasten
G2/M transition. mtp53 diminishes the synthesis of pro-apoptotic proteins (Bax, PUMA, NOXA and
p53AIP1). Repression of these proteins cannot trigger the release of cytochrome c (cyt c) from the
mitochondria. Reduced cytochrome c levels inhibit the activation of caspase 9 (Cas 9) and
subsequently executioner caspase 3 (Cas 3) resulting in the decline of the intrinsic apoptotic
pathway. mtp53 causes reduction in the expression of death receptor, Fas. Diminished Fas
expression is unable to activate caspase 8 (Cas 8) and subsequently executioner caspase 3 and 7
(Cas3&7) resulting in the decline of the extrinsic apoptotic pathway. mtp53 causes repression in the
production of insulin growth factor binding protein 3 (IGFBP-3). Low levels of IGFBP-3 cannot bind
and block insulin-like growth factor 1 and 2 (IGF1&2) anti-apoptotic signals resulting in decline in the
rate of apoptosis. mtp53 inflicts genome instability. mtp53 represses the transcription of
ribonucleotide reductase gene (p53R2) causing reduction in ribonucleotide reductase (RR) synthesis.
This impairs DNA repair. mtp53 represses the Tap63-mediated BubR1 stimulation. Deactivated
BubR1 results in aneuploidy due to chromosome mis-segregation.
 FULL LENGTH APC

15-aa repeat 20-aa repeat Axin binding domain

CtBP-binding region
Oligomerization domain

Armadillo repeat β-catenin binding region EB-1 binding domain

Mutational cluster region Basic region

Binding to Asef1 Binding to β-catenin Binding to CtBP Binding to

EB-1

Binding to Axin Binding to microtubule

C-terminal truncated APC

Binding to Asef1 Binding to β-catenin Binding to CtBP Binding to

EB-1

Binding to Axin Binding to microtubule

Figure 6: The structure of the full-length APC and the C-terminally truncated APC. Full-length APC
has multiple domains to interact with multiple proteins. Majority of cancer-linked mutations of APC
target the mutation cluster region, producing C-terminal truncated APC. Truncated APC loses the
ability to bind with β-catenin, CtBP (C-terminal binding protein), microtubule, Axin and EB-1 (End
binding 1). Adapted from (Hankey et al, 2018).
 CELL PROLIFERATION CELL MIGRATION

mtpAPC mtpAPC

↑ Asef1 EB-1

↑ β-catenin ↓ IQGAP1
↓ CtBP
↑ lamellipodia mDia

↓ Cdc2 Impaired actin

↑ Tcf/Lef Impaired polarization
Microtubule
motility
destabilization
↑ G2/M
Aberrant
↑ c-myc
migration
↑ cyclin D1 Impaired
migration

↑ proliferation

CELL ADHESION CHROMOSOMAL INSTABILITY

mtpAPC mtpAPC

β-catenin-cadherin-actin linkage Microtubule-kinetochore attachment

↓ cell adhesion Chromosome mis-segregation

metastasis Aneuploidy
Figure 7: The various cellular processes affected by mutant APC (mtpAPC). mtAPC drives cell over
proliferation. mtAPC cannot degrade the oncogene β-catenin causing increase in Tcf (T-cell
factor)/Lef (lymphoid enhancer factor) transcription factors-mediated Wnt signalling. mtAPC also
cannot stimulate the C-terminal binding protein (CtBP)-mediated Tcf/Lef transcriptional repression.
Tcf/Lef overstimulation prompt cell growth via c-myc and cyclinD1 gene expression. Excessive Wnt
signalling also can halt the Cdc2 kinase activity causing elevation in the G2/M phase transition.
mtAPC stimulates aberrant migration by over activating Asef1 (adenomatous polyposis coli‐
stimulated GEF 1) leading to excessive lamellipodia formation; thus, impaired cell motility. mtAPC
cannot bind to the IQ-motif-containing GTPase activation protein 1 (IQGAP1) leading to defective
actin-mediated polarized migration. mtAPC inflicts destabilization of microtubules during cell
migration since it cannot bridge EB1 and the formin family protein, mDia. mtAPC can disrupt cell
adhesion. mtAPC impairs the interaction of β-catenin with E-cadherin, γ-catenin, α-catenin and actin
cytoskeleton. Therefore, disrupted cell-cell attachment prompts metastasis. mtAPC causes
chromosomal instability due to impaired interaction between the spindle microtubule and
kinetochore. Thus, the mis-segregation of chromosome during mitosis’ drives aneuploidy.