Oncogenes

 Genes upregulated or dysregulated increase cell growth (cell proliferation,

avoiding apoptosis)
 Distinction between proto-oncogene and oncogene
 Function classification using levels: Growth factors  Growth factor
receptors (tyrosine kinases)  Signal transducers  transcription factors
 Mechanisms of activation:
1. point mutations (HER1, RAS, B-RAF
2. Gene amplification (HER1, HER2
3. Insertional mutagenesis
4. Gene translocations (MYC, ABL  Common in hematologic neoplasms
and sarcomas
5. Protein truncation (HER1, HER2
6. Epigenetic modifications (IGF2 in Hereditary syndromes and sporadic
colorectal cancer, very rare)
 Oncogenes are pleiotropic
Examples of oncogenes
HER family

 Huge family (Her 1 – Her 3), epidermal growth factor receptor

 Tyrosine kinase receptor, on ligand interaction dimerizes  cross
phosphorylation of tyrosine(homo/heterodimer)
 ATP source of kinase phosphate group
 Activates the MAPK cascade and PI3K pathway
 HER1
- EGFR activated by point mutations, protein truncation and amplifications
- Altered in 20% of human cancers [Lung adenocarcinoma, glioblastomas,
and head and neck cancers – typical deletion]
- Mono clonal antibodies 15kD, cetuximab
- Small kinase inhibitors 2kD, mostly ATP analogues
- During diagnosis it’s important to understand intactness of entire
pathway (think RAS)
 HER2 (ERB2)
- Orphan receptor
- Tremendous tyrosine kinase
- HER3 tyrosine kinase receptor that has lost kinase domain during
evolution
- HER2 and HER3 dimerize when HER3 binds its ligand
- While HER2 is active in breast cancer and other epithelial cells,
physiological role is cardiogenesis (division of the heart chambers), there is
a minor homeostatic role conversed for the adult heart, so only minor
cardiotoxicity if inhibited.
- Activation through amplification  homodimerization of HER2 without
regulatory signal  constitutive release of mitogenic signals
- Point mutations and protein truncations
- Important in 20% of breast cancer, amplified in bladder cancers and other
epithelial cancers
- First class therapy is MAB  Trastuzumab. Small kinase inhibitor
 available but less effective
- Response to treatment depends on histochemistry and level gene
amplification
RAS

 RAS is a guanine nucleotide binding protein, GTPase

 K-RAS, H-RAS and N-RAS
 Activates MAPK pathway and downstream of TKR
 In cancer RAS is stuck in activated GTP-bound form
 Activation is usually through point mutations that block GTPase activity
 Hotspot codon are 12, 13 and 61
 Present in about 20% of all human cancers
 K-RAS important in colorectal cancer, lung cancer, pancreatic cancer and
endometrial cancer
 NRAS in 25% of Acute non-Lymphocytic Leukaemia
 First generations drugs tanked
 New hope, potential drug due to G12C mutation
 Patient cured with immunotherapy
B- RAF of RAF family

 A serine-threonine kinase downstream of RAS

 Transmits mitogenic signals
 Activated by point mutation
 20% of human cancers, particularly melanomas
 Effective clinical inhibitors – “rafenib”. Heavily used in clinics
MYC

 Family of transcription factors that dimerize to bind DNA

 Small cell lung cancer, advanced neuroblastoma, Burkitt lymphoma
(EBNA2), acute T cell leukaemia
 Activation is via inter-chromosomal translocation from 8 14, MYC is
attached to strong enhancer and promoter of Ig the heavy chain genes of
B cells
 No drugs yet due to challenge of transcription factors existing with in
nucleus
ABL

 ABL is a non-receptor tyrosine kinase undergoes translocation from

chromosome 9 to 22
 Philadelphia chromosome  BCR-ABL gene  CD 210  Hallmark of
Chronic Myeloid leukaemia
 Present also in Acute lymphoblastic leukaemia
 First treated with Imatinib but constant point mutations have led to
requirement of constant invasion of new generation drugs
BCL2

 BCL family, involved in regulation of apoptosis

  BCL2 involved in the intrinsic pathway and is pro-apoptotic, regulated by
BH3 domain
 Activation is via translocation or overexpression
 Follicular B cell lymphoma or chronic lymphocytic leukaemia
 Therapy via BH3 mimetics like Venetoclax
Clinical implication of oncogenes

 Precision medicine
 Resistance
 Molecular alteration driven therapy vs histotype driven therapy (95% of
therapy is still histotype driven)
Tumor suppressor genes

 Gatekeeper (negative regulators of cell proliferation) vs caretakes

(genome maintenance and DNA repair)
 Gatekeepers (inhibitors of growth factors, signalling and mitogenic
mediators, cell cycle checkpoints, pro-apoptotic genes and genes that
control cell differentiation)  both sporadic and hereditary cancer
 Care takers are DNA damage responders and DNA repair machinery 
hereditary cancers
 Typically require biallelic deletion or inactivation  loss of function.
 Monoallelic  loss of 50% can in some cases be carcinogenic
 Dominant negative mutations
 Promoter methylation  common in neoplasms
 Degraded by viral products e.g. HPV produces E6 ubiquitinates P53 and E7
promotes degradation of RB protein
RB gene

 Grandfather of genes found in retinal tumours

 Essential to control of progress from G1 to S
- E2F is a transcription factor for genes necessary for cell proliferation
- pRB sequesters EF2 and prevents proliferation, CDK phosphorylate pRB to
release E2F
-Under normal conditions there is a cycle of phosphorylation and
dephosphorylations
 Inactivation can be mutations, deletions, stop codons truncations and
degradation of protein e.g. E7 of HPV
 Retinoblastoma, osteosarcoma, small cell lung carcinoma and other
epithelial tumors
 No therapy targeted towards tumor suppressors
 CDK4/6 inhibitors, poor activity in most human cancers but very active in
breast cancer in association with anti-estrogenic drugs
APC

 Important tumor suppressor gene in abdominal cavity (colorectal cancer,

liver cancer etc)
 In physiology:
- WNT (a GF) regulates frizzled receptor
 - In absence of WNT, B-Cat is regulated by degradation by the destruction
complex (APC, Axin, GSK3 and others)  cellular levels of B-Cat are kept
low
- Binding of WNT to frizzled, translocated destruction complex to cell
membrane and block phosphorylation and degradation of B-Cat with the
help of Dishevelled. Increasing B-cat levels.
- B-cat translocates with TCF to nucleus  cell proliferation proteins
 B-cat is bound to E-cadherin, actin and A-cat  link to cell adhesion
 Lack of APC mimics conditions of growth stimulation
 Inactivation via biallelic mutations, haploinsufficiency and methylation
 APC functions also as caretake since it is bound to microtubules and
kinetochores (defective mitosis and genomic instability)
 Inactivated in almost all colorectal cancer. Also, early event in 80% of
human cancers
 No therapy at the moment.
PTEN

 PTEN is a typical gate keeper gene

 PTEN is a PIP3 phosphatase
 PIP3 is involved in the AKT pathway  BAD, mTOR and GSK-beta 
inhibition of apoptosis, cell growth (protein synthesis) and proliferation
 PTEN is antagonistic to PI3K
 Inactivated by haploinsufficiency or epigenetic mechanisms
 Several generations of drugs with numerous isoforms of PI3K enzymes
 Current goal is to find PI3K drugs that are more specific

P53

 Single most important tumor suppressor gene, tetrameric protein

 Guardian of human genome involved in all responses in which there is DNA
damage
 Source and type of DNA damage is not important, any DNA damage
activates ATM and other kinases  Phosphorylate p53 activating it.
Leading to following steps
1. Cell cycle arrest in G1 via p21 WAF1 gene and GADD-45 which inhibits CDK
2. Attempt at DNA repair with help of GADD-45 and others
3. Apoptosis (if DNA repair failed) via BAX and PUMA which inhibit BCL-2
 P53 is controlled by MDM2 (ubiquitin ligase) which functions as negative
feedback
 Consequences of p53 inactivation
- Genetic instability with lots of mutations  drug resistance
- Proliferation instead of dying due to blocked apoptosis
- Tumor heterogeneity
 Inactivation of p53
- dominant negative selection (diagnostic relevance) is an oncogene
- null allele
- Biallelic mutations
  Therapies
- Gene therapy  not used clinically
- Oncolytic virus  not clinically effective
- MDM2 inhibitors  not clinically used
 Peto- paradox  less cancer in large animals than expected due to
multiple copies of p53
Clinical implications for tumor suppressor genes

 Replacement gene therapy (metastases are the problem, can’t be

reached)
 Drugs that skip stop codons (nonsense mutations)
 Synthetic lethality
 Caretakers (except p53) are only active in carcinogenesis  therapy
against them would have no effect clinically
 Caretaker alterations generate antigens
 Caretaker alterations can promote chemotherapy resistance
Hereditary tumor

 Familiarity means the probability of first-degree relatives of cancer patient

developing same type of tumor in comparison to the general public
 First degree relatives frequently have double or more increase in relative
risk
 NB: not always linked to genetic components
 Hereditary oncogenes very rare but common for tumor suppressors
 Hereditary cancer is quite rare (5% of total human cancers)
Feature of hereditary cancer [clinical importance due to use in algorithms]
1. Familial cluster of one tumor type
- High frequency tumor – sporadic and requires more criteria to prove
otherwise
- Rare tumors – hereditary e.g. retinoblastoma 1/100,000 in Bologna
2. Age of onset: younger age of onset in hereditary tumors compared to
sporadic
- Young is relative depending on tumor type
- Retinoblastoma appears average age of onset is 5 years markedly
reduced in relation to sporadic tumors
- Colorectal carcinoma: old change cancer starting from around 60 years,
in hereditary form onset is 40 years
- Breast cancer: sporadic cancer a typical post-menopausal onset,
hereditary breast cancer has premenopausal onset
3. Bilateral or multiple primary tumors of the same histotype
4. Multiple primary tumor of different histotype in same patient
- Patients with breast cancer affected also by ovarian cancer e.g. BRCA1
gene mutation
5. Tumor onset in non-affected sex e.g. male breast cancer
Oncogenes in hereditary cancers
Maybe later
Gatekeeper tumor suppressor genes and hereditary cancer
Retinoblastoma

 First to ever be studied, by Alfred Knudson who hypothesized existence of

Tumor suppressor genes however only identified 30 years later
 Defective RB1 gene
 Two-hit hypothesis [first hit is inherited while second hit is only in tumor
cells and is somatic]
- point mutation
- epigenetic silencing
- insertional deletion
- terminal deletion
 Loss of heterozygosity
 Cancer predisposition vs neoplastic transformation
 Treatment is surgical removal of complete eye bulb but ofc cancer
predisposition persist.
FAP

 Caused by gatekeeper tumor suppressor mutation

 Prevalence of 1:800, very common
 Characterized by presence of numerous colorectal adenomas i.e. polyps
radiological imaging
 Adenoma therefore is benign but still deadly:
- Intestinal obstruction
- Intestinal haemorrhage due to defective angiogenesis
 Haematochezia and melena are common signs of these tumors
 Benign polyps can accumulate mutations and become carcinomas
 Risk of progression is proportional to number of adenomas
 Patients with FAP can also present with duodenal polyps, thyroid
carcinomas and pancreatic carcinomas
 APC gene is altered in FAP and in 80% of sporadic colorectal cancer
 High genomic instability in FAP and colorectal carcinoma linked to activity
of APC
Familial diffuse gastric cancer

 Hereditary mutations in E-cadherin gene, not your typical tumor

suppressor
 E-cadherin is bound (via actin) to B-cat  not only for cell adhesion but
also for proliferation
 Adhesion of epithelial cell important for cell polarity (growth control
mechanism) and proliferation
 Without E-cadherin neighbouring cell growth control mechanism is lost 
uncontrolled proliferation
Von-Hippel-Lindau syndrome (VHL)

 Very rare
  Characterised by presence of hemangioblastoma in retina, spinal cord and
other tumors like renal carcinoma
 VHL is a tumor suppressor that control hypoxic response, important in both
sporadic and hereditary tumors
 Normoxia  Hydroxylated pHIF-1alpha is ubiquitinated by VHL and
degraded
 Hypoxia  No oxygen therefore HIF-1a is not hydroxylated and not
identified and ubiquitinated by VHL. HIF-a binds to HIF-1b and lead to
transcription of hypoxic genes e.g. VEGF, PDGF, EPO etc
 Lack of VHL lead to chronic hypoxic response  transcription of several
growth factors
 VHL is frequently mutated in renal cell carcinoma  role in control of
kidney proliferation
 Highly desirable type drug but still no luck
Caretaker Tumor suppressor genes and hereditary cancer
Importance of DNA repair
1. Direct repair
- Works mainly on lesions caused by methylation (alkylation) of G in to
O6-alkylguanin pairing it with T instead of C  mutation
- Actor is an alkyl transferase  MGMT enzyme
- Alkylating agents are cytotoxic via this system
- Cancer
2. Nucleotide excision repair system
- Active in skin
- Repairs thymidine dimers caused by UV radiation
- DNA cut up and downstream of lesion and replaced via synthesis
- UV light sensitivity and cancer
3. Mismatch repair
- Fixes lesions caused by non-Watson and Crick DNA base pairing 
colorectal carcinoma
- Lynch syndrome
4. Double stranded break repair (DSBR)
- Blunt ends  genome rearrangement
- Error prone
- Tumor cells are frequently mutated at this system
- Can be homologous recombination or non-homologous end joining 
strongly related to breast cancer
- Cancer
5. Crosslink repair
- Endogenous and exogenous agents  inter-strand DNA-DNA links
- Repair is cutting portions up and downstream  double stranded
breaks
- DSBR in involved here to
- Some cancer drugs could cause this
- Defect consequence is cancer and immunodeficiency
Li-Fraumeni syndrome

 Extremely rare type of syndrome – few cases around the world

  Hereditary defect of p53
 Patient develop many different types of cancer
 With-in the same individual development of carcinomas, sarcomas,
leukaemias and lymphomas
 P53 important for development of numerous cancers
Lynch syndrome (Hereditary non-polyposis colorectal carcinoma)

 3% of all colorectal cancer

 Characterized by development of poorly differentiated colon
adenocarcinoma (no previous polyps) at 45 years
 High risk (10-30%) of other carcinomas e.g. endometrial, ovarian, renal,
pancreatic, gastric and bladder
 High penetrance of 85-90%
 Mutations in mismatch repair genes (MHL (50%), MSH2 (35%), MSH6 (10%)
 genomic instability
 Age of onset is 45 years but possible to diagnose early via NGS
 Clinically detected via microsatellite instability
 But mismatch repair and microsatellite occur also in sporadic cancers
 Microsatellite is used to evaluate sensitivity to immunotherapy
Hereditary breast cancer

 Different subtype of breast cancer

- Hormone related type expressing both ER and PR
- HER2 type
- Triple negative subtype (negative for HER2, ER and PR)
 Hereditary breast cancer incidence is 5% of all Hormone related type but
14% of all Triple negative
 BRCA1 and 2 are genes mutated in hereditary breast cancer
 BRCA1 and 2 encode for proteins involved in double stranded repair
protein complexes [BRCA1 in early phase while BRCA2 in later phase]
 Defects in genes leads to cancer and sensitivity to X-rays and other
ionizing radiations
 In 100 families with hereditary breast cancer
- 50 have mutations in BRCA1  Syndrome with female breast cancer
and ovarian cancer
- 1/3 mutations in BRCA2  female and male cancer, along with other
tumors, ovarian less common
- Remainder have no mutation in BRCA1 or 2  BRCAX Could be p53,
ATM and PTEN
 Series of studies have identified alleles associated with high and low risk
 High relative risk  TP53, PTEN, BRCA1 and 2
 Some are still under investigation
 Penetrance
- Probability of expressing a phenotype given a genotype
- Penetrance of hereditary breast cancer is 40-80% (very broad range) 
highly variable from one family to another
- Penetrance can be considered in terms of cumulative risk as a function
of age
 - BRCA1 High risk families’ increases steeply with age  cumulative risk
at 70 years is 80-90% for breast cancer and 60% ovarian
- BRCA2 families at 70 have a cumulative risk 80-90% for breast cancer
but only 20% ovarian cancer
- However, considering relatives of unselected cancer patients’ risk is
60% BRCA1 and 40% BRCA2

Tumor progression

 Malignant tumors are a result of several mutations and genetic alterations

occurring over a long time
 Hits  sequence of events
 Tumor progression is incremental accumulation of genetic damage 
hyperplasia (preneoplastic)  benign tumor  malignant  invasive 
metastatic
 Minimum number of mutations is 2 e.g. Retinoblastoma (very early onset)
but other cancer e.g. lung require 20 hits  longer span which gives
opportunity for prevention
 Natural history is timeline of events of progression. Only studied for
carcinomas and leukaemias but not sarcomas
Natural history
Clonality

 Use of G6P dehydrogenase in women with leiomyosarcoma to assess

whether tumour are monoclonal  expression of G6PD in normal tissue is
a mix 50/50 of different isoforms but with in a tumor all cells express only
one isoform of G6PD
 Other studies in T and B lymphocytes in multiple myeloma  all expressed
antibodies derived from single plasma cells
 Most human tumors are indeed monoclonal
 Polyclonal tumors exist e.g. retinoblastoma [could be single cell origin or
confluence of 2 different tumors) or in powerful carcinogens  multiple
clones
 Field carcinogenesis natural history of mixed tumors, polyclonal in origin
to asses field of cells and factors needed for multiple tumors to fuse e.g.
polyps in colorectal cancer and alcohol or smoking and multiple oral
lesions
 Clonal evolution