Molecular Perspectives in Oncology Development of Cancer: A Multistage Process

Dra. Mari-Ann Bringas - Successive stages of:

o Mutation – affect regulatory pathways that control cell
Learning Objectives communication and growth
- Differentiate between normal and cancer cells o Competition with a proliferative growth advantage –
- Understand basic characteristics of cancer at the biochemical and cell transformed to a state of uncontrolled growth
molecular level (immortalization)
- Identify gene families involved in carcinogenesis o Selection – individual cells prosper at the expense of
- Understand the mechanisms that lead to cancer formation other cells in the body
- To understand processes of chemical, physical and viral - Benign tumour or neoplasm – not acquired invasive properties
carcinogenesis - Malignant cancer – invasive: infiltrates into surrounding tissues
- To understand the different pathways of malignancy and their o Reach lymphatics  metastasizes to other parts of thy
components body
- To identify potential targets for development of cancer
therapeutics Characteristics of Cancer
- Clonality, Autonomy, Anaplasia, Metastasis
Cancer
- Nowell’s Law: Clonality
o Cancer is an overgrowth of cells bearing cumulative - Cancer is a genetic disease (cellular level)
genetic injuries that confer growth advantage over the o Tumours arise as clones from a single cell
normal cells o The development of the malignant clone is due to
- Robin’s definition: mutations in DNA
o Cancer cells can be characterized as antisocial, fairly o Mutations may be due to:
autonomous units that appear to be indifferent to the  Random replication errors
constraints and regulatory signals imposed on normal  Exposure to carcinogens
cells  Faulty DNA repair process
- Genetic mutations – critical role in the development of cancer
CANCER CELLS VS NORMAL CELLS o Dominant mutation
Cancer Normal Cells  Conversion of proto-oncogenes to
Loss of contact inhibition Presence of contact inhibition oncogenes
Increase in growth factor secretion Intermittent or coordinated growth  First discovered as ‘oncogenes’ in
factor secretion transducing retroviruses
Increase in oncogene expression Oncogene expression is rare o Recessive mutation
Loss of tumour suppressor genes Presence of tumour suppressor  Damage or loss of tumour suppressor genes
genes  First discovered as ‘inhibitors of
transformation’ in cell fusion experiments
Stages of Cancer Progression or as genetic mutations (chromosomal
- Normal (Primary Cells) deletions) that caused cancer
o Culture divides for several divisions (retinoblastoma: Rb gene)
o Growth of cells is restricted by properties of individual  Also identified as cell proteins complexed
tissue with IE/E proteins of DNA viruses (SV40,
- Benign (Immortalization) Adenovirus): e.g. p53
o Crisis – majority of cells die, a few cells grow out - Consequences of genetic instability:
o Immortalization – established cell line divides o Phenotypic heterogeneity
indefinitely but cells continue to adhere to substratum, o Tumour progression
require serum, and are inhibited by contact
- Cancerous (Transformation) Autonomy
o Transformed cells are independent of anchorage, - Cancer cells are able to proliferate
serum, contact inhibition o Despite the signal of regulatory influences
o These cells also change shape, round up and grow into o DEPENDENT ON THE CELL CYCLE
a focus - Unrestricted proliferation
o Transformed cells may also form a solid tumor o Usually leads to tumour formation
- Systemic Spread (Metastasis) - Mechanisms of autonomy
o Now fully tumourigenic, cells become mobile and can o Growth factor secretion
migrate to start new colonies o Increased number of receptors
o Tumours form at new locations o Independent activation of key biochemical processes
o
The Cell Cycle G0: reactivation

G0: indefinite
withdrawal

Regulation of Cyclins and Cdks
Check Points
- Restriction point
o Pause in response to environmental cues
o Passing this point means the cell is committed to
complete the rest of the cell cycle and division
o Wait for molecular signals
- Regulatory signals:
o Stimulators of cell growth  GO! (proto-oncogenes)
o Inhibitors of cell growth  STOP! (tumour suppressor
genes)
- Cancer formation
o Stimulatory gene product  hyperactive
o Inhibitory gene product  inactive
Cell Cycle Regulation
- Process assures that the cell accurately duplicates its contents
- Important checkpoints at G1 and G2 are regulated by protein
kinases / cyclins (cdk – cyclin dependent kinases)
- Checkpoints determine whether the cell proceeds to the next
phase of the cycle or not
G2 / M Checkpoint
- Regulated by cyclin B/cdc2 (mitosis promoting factor)
- Activity of the cyclin with its substrate results in:
o Chromosome condensation
o Nuclear breakdown
o Spindle formation
G1 / S Checkpoint
- Area most often disrupted in cancer
- Mechanism of regulation is complex – phosphorylation of pRB
gene results in:
o Activation of several genes needed for S-phase
progression
o Promotes differentiation through association with
transcription factors
Retinoblastoma Gene Activation
Cyclin Regulators
- Regulated by cdk inhibitors (cdki)
- May be induced by growth inhibitors and inhibited by positive
growth factors
- Genetic alterations in cdki occur with high frequency in some
cancers
- P21
o Inhibits cell cycle progression
o Permits DNA repair to take place
- P53 – “guardian of the genome”
o Halts cell cycle progression to facilitate DNA repair
o In cases of severe DNA damage, activates apoptosis
o Mutations in p53 – most common genetic alterations
found in human cancer
Anaplasia
- Loss of differentiated function
- Bizarre-looking cells
- Large nuclei, prominent nucleoli, increased chromatin
- Increased and/or abnormal mitosis
- Aneuploidy
- Partial or complete loss of normal architecture
Invasion and Metastasis
- Defining characteristic of malignancy
- Invasion – active translocation of neoplastic cells across tissue
barriers
- Critical pathologic point:
o Local invasion
o Neo-vascularization
- May occur before clinical detection
Attributes of Cancer  Loss of function results in unregulated
- Basic steps in metastasis: growth
o Destruction of basement membrane o Mutator genes
o Attachment to the laminin of the distant BM  Loss of function results to increased rate of
- Good metastasizers: up-regulated genes mutations
o EDGF receptor
o Basic fibroblast growth factor Oncogene Family
o Type IV collagenase - Oncogenes
o E-Cathepsin (under-expressed) o Promote cell differentiation
o Cathepsine B (a lamininase) o Dominant and highly-conserved
o Heparanase - Types of oncogenes:
- The process of metastasis involves these steps: o V-oncs (viral oncogenes)
o Escape from the primary tumour to the circulation o C-oncs (cellular oncogenes)
(intravasation) - Protooncogenes  mutation  oncogenes
o Survival in the circulation - Classification of oncogenes:
o Arrest at a new site Secreted growth factors C-sis, hst
o Escape from the circulation (extravasation) Cell surface receptors Erb B, fms, ret, trk, fes
o Migration into the interstitial space at the new site and Intracellular transducers (components of signal C-src, c-abl, mst, ras
initiation of growth transduction pathway)
o Initiation of new vessel formation (angiogenesis) to DNA-binding nuclear proteins Myc, jun, fos
provide nutrients to growing tumour mass Regulators of the cell cycle Bcl, bax, bad

Signal Transduction

Oncogene Family: Mechanisms of Oncogene Activation

- Point Mutation
Angiogenesis
H-ras [codon 12]
Normal CGC  Gly
Bladder ca CTC  Val
H-ras binding with GTP results in perpetual cell division
- Gene Amplification
o Double minutes
o HSR’s (homogenously staining regions)
o Normal copy  multiple copies
- Gene Translocation
o Burkitt’s Lymphoma

Triad of Invasion
- Adhesion with the basement membrane
- Local proteolysis
- Mobility – ability to translocate through dents in body’s tissue
barriers

Molecular Carcinogenesis
- Mutation – molecular hallmark of most forms of cancer
- Gene families in cancer development:
o Oncogenes
 Normally promote normal cell growth
 Mutations convert them to oncogenes
o Tumour suppressor genes
 Normally restrain cell growth
 o Chronic Myelogenous Leukemia (CML) - Retinoblastoma Gene (RB gene)
o Rare form of childhood malignancy
o Forms: hereditary or sporadic
o pRB (protein RB)
 105-kDA nuclear protein
 Inhibits E2F (prevents G1  S transition)
o Inhibited by:
 Phosphorylation
 Viral oncoproteins (E1A, HPV E7)
o Rb gene activation:

- Viral Gene Integration - Tp53 Gene

o Normal individuals synthesize fast promoter regions o Location: 17p13.1
o Viral oncogenes inserted into the genome will increase o Product: p53 protein (53 kDA)
the rate of expression of the abnormal gene o Function: induction of DNA repair or apoptosis
o Mutation: point mutation  deletion (results to loss of
Tumour Suppressor Gene Family function and extended lifespan of p53)
- TS Gene Family o Clinical conditions:
o Inhibits growth and multiplication of mutated cells  Carcinomas
o Prevents neoplastic transformation  Li-Fraumeni syndrome
o Recessive and highly conserved o Inhibited by:
o Classification of TS genes:  FB1, HPV E6, mdm2 point mutation
 Cell Adhesion Molecules (CAM)
 Regulators of the cell cycle (RB1, Tp53) Activation of p53 can
o Knudson’s Two-Hit Hypothesis

st
1 hit: TS mutation or inherited mutation have 2 results: cell cycle
nd
 2 hit: Gross chromosomal loss arrest or apoptosis
 A cell can initiate a tumour only when it
contains TWO mutant alleles. A person who
inherits a mutant allele must experience a
second somatic mutation to initiate
tumourigenesis. Persons with inherited
mutations frequently develop cancer in
more than one site.
Inhibition of Apoptosis Occupational Risk Factors
- Adenovirus, papovirus, papillomavirus proteins INACTIVATE p53 ETIOLOGY: SITE OF MALIGNANCY:
- P53 induces cells into arrest or apoptosis by transcriptional Arsenic Lung, skin, liver
activation of specific control genes and other mechanisms (not Asbestos Mesothelium, lung
fully understood) Benzene Leukemia
- P53 is the most frequently mutated gene in human tumours Benzedine Bladder
Chromium compounds Lung
Radiation (mining) Numerous locations
Mutator Gene Family
Mustard gas Lung
- Involved in ensuring the fidelity of replication
Polycyclic hydrocarbon Lung, skin
- Function:
Vinyl chloride Angiosarcoma of liver
o Checks for and corrects mismatched pairs
- Mutation:
Carcinogen Metabolism
o Inefficient repair and replication leading to propensity
- Three main categories:
of oncogenes and tumour suppressor gene defects
o Chemical carcinogens
o Leads to the formation of Micro-Satellite Instability
o Physical carcinogens
(MSI)
o Viral agents
- Carcinogens  mutations due to environmental factors 
Recap of Oncogenes
CANCER
- Protooncogene  gain of function  cancer
- TS gene  loss of function  cancer
Chemical Carcinogenesis
- Mutator gene  loss of function  cancer
- Initiation (primary exposure)  promotion (transformation) 
progression (cancer growth)  frank cancer
Carcinogenesis
- Initiation
- Occupation-related causes
o Normal cells are exposed to a carcinogen
- Lifestyle related
 Direct-acting carcinogens
o Smoking / alcohol
 Indirect-acting carcinogens
 Tobacco-related  lung, pancreatic,
o Not enough to cause malignant transformation
bladder and renal cancer
o Requires one round of cell division
 Tobacco + alcohol  oral cavity and
o Pro-carcinogen  cytochrome-p450  ultimate
esophageal cancer
carcinogen
 Tobacco + asbestos / mining / uranium /
- Promotion
radium  respiratory tract and lung cancer
o Initiated cells are exposed to promoters
o Sexual practices
o Promoters are NOT carcinogens
 Sexual promiscuity, multiple partners,
o Properties of promoters:
unsafe sex, Human Papilloma Virus (HPV) 
 Reversible
anogenital and cervical cancer
 Dose-dependent
o Diet
 Time-dependent
 Nitrates, salt, low vitamin A, C, E, low
o Types of carcinogens:
yellow-green vegetables  gastric and
 Direct
esophageal cancer
 Pro-carcinogens  ultimate carcinogens
 High fat, low fiber, low calcium, high fried
foods  colon, pancreatic, prostate, breast DIRECT ACTING: PRO-CARCINOGENS: PROMOTERS:
and uterine cancer Cyclophosphamide PAHs Saccharine
 Mycotoxins  liver cancer Chlorambucil Aromatic amine Cyclamates
- Ionizing radiation (x-rays) Busulfan Aromatic dyes Estrogen
- Multi-factorial causes Melphalan Aflatoxin B1 Diethylstilbestrol
- Viral carcinogens Nitrosamines
- Chemical carcinogens Asbestos
Chromium, nickel
Sources of Free Radicals Metals
- X-rays, stress, toxins, sunlight, solvents, pollution, cigarette, Arsenic
pesticides, herbicides, medications, airline travel, radioactivity,
food additives, polluted foods, high heat cooking, synthetic Physical Carcinogenesis
materials, household cleaners, environmental chemicals - Radiation
- Smoking: 10 quad trillion free radicals per cigarette o Sunlight
o Artificial sources of UV light
 UV A = 320-400 nm
 UV B = 280-320 nm
 Inhibition of cell division
 Inactivation of enzymes
 Induction of mutations
 Cell death at high doses
 May cause squamous cell Ca,
basal cell Ca or
melanocarcinoma
 UV C = 200-280 nm
 Filtered by ozone
o X-rays
o Radio-chemicals
 o Nuclear fission o E7 induces Rb degradation by proteosomes
- Ionizing Radiation - Human Papillomavirus (HPV types 16, 18, 31, 33 & 35)
o Electromagnetic rays o Interruption of the E1/E2 ORF  E2 is not expressed
o Particulate matter  over-expression of E6 & E7
o Mechanism: - Epstein-Barr Virus (EBV)
 Increase free radicals and mutations o In Burkitt’s, B-cell, Hodgkins lymphomas, NP Ca
o Pathology: o Tropism:
 Leukemia > thyroid Ca > lung and breast Ca  CD21 + cells (B-cells, epithelial cells)
o Resistant tissue: o Mechanism:
 Bone, skin, GIT  Viral entry  episomal existence -> latency
 + LMP-1, EBNA-1, EBNA-2 
Viral Oncogenesis immortalization
- Hepatitis B Virus (HBV)
o Induction of chronic hepatocyte injury (+) HBx
o HBx activated protein kinase C for transformation

Viral Agents (RNA Viruses)

- Human T-cell Leukemia Virus (HTLV)
o A retrovirus
o Tropism: CD4 + cells

- Three types of oncogenic retroviruses:

o Rous sarcoma virus – a transducing retrovirus
o Murine leukemia virus – a slow (cis)-acting
nontransducing retrovirus
o Human T-cell leukemia virus – a long-latency
retrovirus

COMPARISON OF DNA VIRUSES AND RETROVIRUSES

DNA VIRUS RNA VIRUS
SV 40, adenovirus, papillomavirus Retroviruses
Normally lytic Not lytic (infection does not lead to
cell death)
Integration NOT an essential part of Integration is an essential part of
viral multiplication cycle viral multiplication cycle
- Viruses may kill or transform cells Oncogenes are unique viral proteins Oncogenes are NOT unique gene
o Productive infection in permissive cells: (T Ag, E1A/E1B, E6/E7) products: no role in viral life cycle
 Early infection – entry and production of Transform cell: INACTIVATE Transform cells: STIMULATE
virus components INHIBITORS of cell cycle (tumour ACTIVATORS of cell cycle (proto-
 Late infection – replication and assembly of suppressor genes) oncogenes)
particles Recessive mutation Dominant mutation
 Lysis – cell death and release of viruses
o Transformation in non-permissive cells Principal Pathways of Malignancy
 Abortive infection – virus enters - Proliferation
 Integration – viral DNA becomes part of - Cell-cycle progression
host genome - DNA repair
 Transformation – cells change shape and - Immortalization
grow - Apoptosis
- Metastasis and invasion
Viral Agents: DNA Viruses
- Adenovirus, Papovirus and Papillomavirus proteins actively Growth Factor Signalling Pathway
disrupt Rb-E2F complexes - Uncontrolled and uncoordinated proliferation
o RB (hypophosphorylated) bound to histone - Uncontrolled growth stimulated by:
deacetylases (Hdac) and E2F represses transcription o Increased secretion of growth factors
from E2F responsive promoters o Increased growth factor secretion
o SV40 LT, Ad E1A, HPV E7 bind Rb o Independent activation of certain enzyme and protein
o Hyperphosphorylated Rb are not bound production pathways
 E2F:Dp transcriptionally active - Growth factors:
o LT, E1A: sequester hypo-Rb (and related p107, p130), o PDGF, EGF, FGF, VEGF, NGF
and disrupt Rb-E2F complexes (LT may recruit - Receptor Tyrosine Kinase Pathway (RTK) – main pathway
chaperone Hsc70) o RTK ligands: PDGF, EGF, FGF, VEGF, NGF
 o Functions of RTK: DNA Repair Pathways
 Promotion of cell survival - Cancer – “malady of genes”
 Regulation of cell proliferation and - Defects in the maintenance of genome stability
differentiation - Repair mechanisms:
 Modulation of cellular metabolism o Mismatched excision repair
o Base excision repair
Proliferation o Nucleotide excision repair
- Therapeutic implications: o Double strand base repair
o Blocking of GF mitogenic signalling achieved by: - Clinical significance: HNPCC – Hereditary Non-Polyposis Colonic
 Preventing binding of GF to receptor or Cancer
receptor dimerization with specific agent o Mutations in genes involved in DNA repair pathway
 Preventing receptor activation with small (MSH1, MSH2)
molecule inhibitors o Features:
 Blocking cytoplasmic proteins downstream  Somatic defects in repeated DNA elements
of the activated receptor pathway leading to microsatellite instability (MSI)
 Inactivation of TGF-B (tumour suppressor)
Cell Cycle Regulation  Inactivation of BAX gene
- Process assures that cell accurately duplicates its contents
- Important checkpoints at G1 and G2 are regulated by protein Immortalization
kinases / cyclins (cdk) - Telomeres – specialized structures at chromosome ends
- Checkpoints determine whether the cell proceeds to next phase generated and maintained by telomerase
of the cycle - Telomerase – ribonucleoprotein enzyme which preserves the
- Cyclin regulators: integrity of telomeres. Also a key component in immortalization
o P21 of cancer cells
 Activated by p53 - Telomere length – represents molecular clock that determines
 Inhibiting the cell cycle progression and the lifespan of the cell
permitting DNA repair to take place - Clinical significance:
o P53 o Most normal adult tissues have NO telomerase activity
 Guardian of the genome o Telomerase activity is present in 90% of tumours
 In the presence of DNA damage: - Therapeutic implication:
 Halts cell cycle progression to o hTERT – protein identified to be catalytic subunit of
facilitate DNA repair telomerase
 In cases of severe DNA damage, o Limiting component of telomerase activity
activates apoptosis o Can be a target for small molecule inhibitor
 Mutations: most common genetic
alterations found in human cancer Apoptosis
- Cyclin inhibitors: - Programmed cell death
o CDK inhibitors – inhibit the activity of CDK / cyclin - Importance:
complex o Steady-state kinetics of normal tissues
o 2 groups: o Focal deletion of cell during normal embryonic
 INK4 family – p15, p16, p18, p19 development
 CIP-KIP family – p21, p27 o Seen after chemotherapy or radiation
o Actions: - Balance between proliferation and apoptosis is critical in
P15 Change response to anti-mitogenic agents determining growth or regression
P16 Inhibits CDK-cyclin D
P19 Induces p53 stabilization Components of Apoptosis
P21 Induces cell cycle arrest via activation by p53 - Caspases
P27 Inhibits CDK2 / cyclin E o Caspases – Cysteine-containing Aspartate Specific
Proteases)
Cell Cycle Progression o Initiator caspases – activated in response to cell death
signal
- Clinical significance: Oncogenic alterations in cell cycle regulators
o Loss of p53 and pRB function as tumour suppressors o Executor caspases – progress the death signal
o Increased expression of Cyclin D1 (mantle cell activating cascade resulting to DNA fragmentation and
lymphoma) cell death
o CDK4 amplification in sarcomas, gliomas o Components:
o Mutations in p16-binding domain of CDK4 (familial  Caspase prodomains – DED, CARD
melanoma)  Death ligands – TNF, Fas, TRAIL
o Inactivation of INK4  Survival signals – NFK-beta
o Alterations in cyclin D1, p16 - Cytochrome C
o Decreased levels of p27 (breast Ca) o Component of mitochondria released in response to
o Overexpression of cdc25 apoptotic signals
- Therapeutic implications: approaches using inhibitors of CDKs as - BCL-2 Family
therapeutic agents o Located upstream in the pathway
o Small molecules o Provides decisional checkpoint in the fate of cell after a
o Protein therapy death stimulus
o Antisense o Contains BH1-BH4 domains necessary for interaction
- Anti-apoptotic signals: BCL-2, BCL-XL
o Gene therapy
- Most cytotoxic agents block the cell cycle in S / G2 / M phase - Pro-apoptotic signals: BAX, BAD, BAK, BID
Apoptotic Pathways
- FAS-mediated pathways
o FAS – cell surface receptor of TNF family which binds
to FAS-L
o Eliminates unwanted activated T-cells
o Pathway for cytotoxic-mediated signalling
- P53-mediated apoptosis
o Important after chemotherapy and radiation
o Induction of BAX and down-regulation of BCL-2
o Induced expression of FAS and DR5
Evading Apoptosis
- Therapeutic implications
o Antisense oligonucleotide against BCL-2 in the
treatment of lymphoma
o BCL-2 antisense as chemo-sensitizing agent in solid
tumours
o TRAIL (TNF-Related Apoptosis Inducing Ligand) to
induce apoptosis
Significance of Apoptosis
- Clinical significance: tumour angiogenesis switch is triggered as a
result of shift in the balance of stimulators to inhibitors
- Therapeutic implications:
o Metalloproteinase inhibitors (MMPI) – block the
degradation of basement membrane
o Inhibitors of endothelial function – thalidomide, TNP
470, endostatin
o Anti-angiogenic factors – tyrosine kinase inhibitors
(VEGF, FGF, PDGF)
o Interferon – angiogenic inhibitor
o COX-2 inhibitor – thromboxane-A2 as critical
intermediary of angiogenesis
Additional Resources...

Angiogenesis Components
- Endothelial cells
- Inducers of angiogenesis
o VEGF – main inducer
o TGF-beta
o TNF-alpha
 Low concentration – inducer
 High concentration – inhibitor
o PDGF / thymidine phosphorylase
o TGF-alpha
o EGF
o IL-8
- Cell Adhesion Molecules (CAM)
o Mediate cell-cell adhesion processes
o Selectins
o IG supergene family – ICAM, VCAM
o Cadherins
o Integrins – vitronectin receptor
- Proteases
o Degrade ECM to provide suitable environment for EC
migration thru adjacent stroma
 E.g. metalloproteinases (MMP)
- Inhibitors of angiogenesis
o Interferon
o TSP-1
o Angiostatin
o Endostatin
o Vasostatin

Prepared by: Paolo Warren (Med-IA)