Tumorgenes Flashcards
Multistep tumorigenesis
The accumulationof mutations promotes tumor progression:
normal -> initiated -> pre-cancer (mild -> moderate -> severe -> cis) -> cancer
Novel capabilities acquired during tumor development:
* self-sufficiency in growth signals
* Insensitivity to growth-inhibitory (antigrowth) signals
* evasion of programmed cell death (apoptosis)
* limitless replicative-potential
* sustained angiogenesis
* tissue invasion and metastasis
Mutations
Somatic mutations
* Most changes in DNA usually happen during our lifetime
Germline mutations
* Some people inherit DNA mutations from their parents
* that greatly increase their risk for developing certain cancer
Driver mutations
* Causally implicated in oncogenesis
* Confer growth advantage on the cancer cells
Passenger mutations
* Without functional consequences for oncogenesis
* No growth advantage on the cancer cell
Oncogenes vs Tumor suppressor genes
Most cancers are caused by mutations to two basic classes of genes: proto-oncogenes and tumor suppressor genes
Proto-oncogenes
* code for proteins that stimulate the cell cycle and promote cell growth and proliferation
Tumor suppressor genes
* code for proteins that repress cell cycle progression and promote apoptosis
An important difference between oncogenes and tumor suppressor genes:
* oncogenes result from the activation (turning on) of proto-oncogenes
* tumor suppressor genes cause cancer when they are inactivated (turned off)
Oncogenes
Proto-oncogene
* is a normal gene that can become an oncogene due to mutations or increased expression
* encodes for proteins that help to regulate proliferation, differentiation, and cell survival
* often involves in signal transduction
Oncogene
* a gene that can transform cells
* help tumor cells to survive and proliferate
Examples
- Growth factors, growth factor receptors (e.g. receptor tyrosine kinase HER2)
- Cytoplasmic signal mediators (e.g. kinases, Ras protein)
- Nuclear signal mediators (e.g. transcription factors, e.g. Myc)
- Regulator proteins of the cell cycle (cyclines, cyclin-dependent kinases)
Conversions of photo-oncogenes to oncogenes
Mutation in the proto-oncogene
Changes in the protein structure causes
* permanent activation of oncoproteins
* a loss of regulation
Protein concentration
Increased gene expression caused by
* an increase of protein expression (through misregulation)
* an increase of protein (mRNA) stability, prolonging its existence and thus its activity in the cell
* gene duplication (one type of chromosome abnormality), resulting in an increased amount of protein in the cell
Chromosomal translocation
* increased gene expression
* formation of hybrid genes and fusion proteins e.g. Philadelphia chromosome -> chronic myeloid leukemia
HER2: a proto-oncogenic receptor
- Member of the epidermal growth factor receptor (EGFR) family
- Membrane-bound receptor tyrosine kinases
- Formation of homo- and heterodimers
- manifold cellular functions: proliferation, differentiation, survival, apoptosis, cell adhesion and mobility
- HER2: Orphan receptor
- preferred dimerization partner for all other HER receptors
- HER2 is frequently over expressed in cancer of the breast, ovarian, prostate and stomach
- amplification of Her2 encoding gene in ca 15-30% of all breast cancer
- HER2 overexpression leads to strong and constant proliferative signaling
Breast cancer
- Inherited disease (5-10%) e.g. BRCA1, BRCA2
- Sporadic disease (90-95%)
*Invasive ductal carcinoma ~ 80-90% of all breast cancer diseases
*Invasive lobular carcinoma (rare) - increase also the risk of ovarian, colorectal, and prostate cancers
Micro-anatomy of the milk duct
A milk duct
* is lined by epithelial cells (dark purple nuclei) and
* surrounded by mesenchymal tissue (stroma)
containing connective tissue cells (e.g. fibroblasts and adipocytes) and collagen matrix (pink).
The ductal system is lined by 2 cell layers
1. luminal glandular epithelial layer (LE; grey)
2. myoepithelial cell layer (ME; brown) lying between LE and the basement membrane (= basal cell layer)
Calponin is a marker of my-epithelial cells.
Tissue architecture becomes abnormal in tumors.
- Mildly hyperplastic milk ducts (atypical ductal hyperplasia, ADH)
- Ductal carcinoma in situ (DCIS) (precancerous lesion)
- Invasive ductal carcinoma (IDC)
Intertumor heterogeneity of ductal carcinoma
LUMINAL A
Typical status of ER/PR/HER2 and others: ER+ and/or PR+, Ki67 low*, HER2-
Prevalence: 42-59%
Notes: most common and best prognosis
LUMINAL B
Typical status of ER/PR/HER2 and others: ER+ and/or PR+, Ki67 high*, HER2- or HER2+
Prevalence: 6-19%
Notes: Compared to LumA: poorer prognosis
HER2+
Typical status of ER/PR/HER2 and others: ER-, PR-, HER2+
Prevalence: 14-20 %
Notes: often poor prognosis
BASAL-LIKE/TRIPLE-NEGATIVE (TN)
typical status of ER/PR/HER2 and others: Markers of basal cytokeratins (CK4/5, 15, 17), typically do not express ER, PR, HER2 (TN)
Prevalence: 7-12%
Notes: often aggressive, poorer prognosis
*Levels of Ki67 (proliferation marker): low: <14% cells positive, high: >14% cells positive
Her2 as prognostic and therapy selection marker
Routine evaluation of the HER2 status by FDA-approved assays
- immunohistochemistry and fluorescence in situ hybridization (FISH)-
HER2/neu Immunhistochemie:
Score: 0 = negativ
Score: 1+ = negativ
Score: 2+ = schwach positiv -> HER2/neu FISH -> Positiv = Trastuzumab Therapie -> Negativ = keine Trastuzumab Therapie
Score: 3+ = stark positiv -> Trastuzumab-Therapie
Trastuzumab:
recombinant, humanized monoclonal antibody directed against the extracellular domain
of the HER2 protein (chimeric AB)
Mouse paratope (antigen binding site)
Human effector region (constant domains)
FDA: Food and Drug Administration
Immunohistochemical analysis of HER2
The semiquantitative scoring system is based on intensity and extension of the staining.
(A) Negative: no membrane staining or <10% of cells stained.
(B) 1+: incomplete and weak membrane staining in >10% of the cells.
(C) 2+: weak to moderate complete membrane staining in >10% of the cells. -> FISH
(D) 3+: strong and complete membrane staining in >10% of the cells.
Detection of chromosomal abnormalities
Fluorescence in situ hybridization (FISH)
Probe labelling by e.g. PCR amplification with fluorophore-modified nucleotides
-> * FDA-approved DNA Probe Kits for clinical diagnostic
Interphase or metaphase nuclei from formalin-fixed, paraffin-embedded human tissue specimens -> Denaturation of the probe and the target DNA -> Hybridization of the Probe-target molecules
Dual color FISH assay to test HER2 amplification
- Determination of the HER-2/CEP 17 ratio
* Counting the signals for Her2 and CEP17 in the same 20 nuclei
* Dividing the total number of Her2 signals by the total number of CEP17 signals - Determination of the mean Her2 signal number per cell in case of <2
ISH-negative: <4,0
ISH-positive: ≥6,0
ISH-equivocal: ≥4,0 - <6,0
Fluorophore-labelled DNA probes:
Red: specific for the Her2 gene locus
Green: CEP17: specific for the satellite DNA sequence at the centromeric region
CEP17 probe acts as an internal control and corrects for polysomy (gene amplification versus chromosome 17 polysomy)
CEP: chromosome enumeration probe
Blue: Nuclei counterstaining with DAPI
Non-amplified Her2: Her2 (red)/CEP17 (green) <2
Amplified Her2: increased numbers of Her2 gene signals (red), Her2/CEP17 ratio: >= 2
Treatment of HER+ patients
Therapeutic effects:
TRASTUZUMAB (HERCEPTIN)
* Activation of an immune response (ADCC (antibody-dependent cell-mediated cytotoxicity) by NK cells)
Other possible mechanisms of action
* Increasing the rate of degradation of HER2
* Prevents downstream tumorigenic signaling leading to cell cycle arrest and apoptosis
Major side effect: cardiac dysregulation
LAPATINIB (TYVERB)
(small-molecule, reversible tyrosine kinase inhibitor)
* Binds to the ATP-binding pocket of HER1 and HER2
* Prevents phosphorylation of the kinase domain
* Prevents downstream tumorigenic signaling leading to cell cycle arrest and apoptosis
Major side effect: affect liver function
Invasive ductal breast cancers with brain metastasis
* Combined administration of Lapatinib & Trastuzumab (Lapatinib can pass the blood–brain barrier)
HER2 somatic mutations
Pitfalls: HER2 somatic mutations in HER2 gene amplification negative breast cancer
Homo- and heterodimerization (HER2)2; HER2/HERX -> Activation of downstream signal transduction pathways which can differ in respect to signal strength and pathways
Tumor suppressor genes
Functions:
1.) Repression of genes that are essential for continuing the progression of the cell cycle
2.) Coupling the cell cycle to DNA damage
3.) If damage cannot be repaired, the cell should initiate apoptosis
4.) Proteins that function in DNA repair, preventing cells from replicating mutations
5.) Some proteins involved in cell adhesion prevent tumor cells from dispersing, block loss of contact inhibition and inhibit metastasis
Some examples:
pRb (retinoblastoma) -> proliferation
APC (colorectal cancer) -> proliferation
p53 (many tumors) -> division, apoptosis
BRCA1,2 (breast cancer) -> DNA repair
Tumor suppressor genes -> Gatekeeper, Caretaker, Landscaper
Gatekeeper
Genes that directly hinder cell division or promote cell differentiation or cell death
* Mutations lead to the appearance of unregulated dividing cells (e.g. pRB in Retinoblastoma)
Caretaker
Genes that encode proteins responsible for maintaining the integrity of the genome
Inactivation of a caretaker gene
* does not promote tumor initiation directly
* but leads to genomic instability that increase the frequency of mutations (e.g. BRCA1 and 2 (repair of DNA breaks) in breast cancer)
Landscaper
Genes that encode products that help create environments that control cell growth
* Regulation of extracellular matrix proteins, cellular surface markers, cellular adhesion molecules and growth factors (e.g. PTEN)
BRCA: Breast Cancer; pRB: Retino blastoma protein
The prototype TSG: Retinoblastoma protein
° pRB is encoded by the Rb1 gene
° Nuclear pRB (928 amino acids, 110 kDa)
° Function:
- suppression of cell division in absence of mitotic signals
- preventing transition from G1 phase to S phase
° E2F1-3: transcription factors (TF)
° Regulation of pRB activity by phosporylation:
° pRB hypophosphorylation: binding and inhibition of E2Fs
° pRb hyperphosphorylation by activated CDKs: release of E2Fs which will be active
° Transactivation of E2F-target genes facilitates the G1/S transition and S-phase
° In cancer cells, disrupted pRB function results in aberrant cell proliferation
Loss of Retinoblastoma protein
- Initiating event in hereditary as well as sporadic retinoblastoma
- Increased risk of patients with hereditary retinoblastoma to develop other cancer types (e.g. osteosarcoma, small-cell lung carcinoma)
- Alteration of the Rb gene in several sporadic cancer types are associated with progression
- genetic mutation
- viral inactivation
- phosphorylation
- degradation
Two forms of retinoblastoma
FAMILIAR (10%) OR SPORADIC BILATERAL (30%)
* Germline mutation & de novo germline mutation (sporadic!)*1
* Bilateral: in both eyes
* Multifocal: Multiple tumors in each eye
* After treatment:
Increased risk of
- Osteosarcoma (>400 times above normal), small cell lung carcinoma & melanoma
*1 De novo germline mutation
° No familial history but the same characteristics as the familial disease
° Mutations often in sperm (high proliferation rate)
SPORADIC UNILATERAL (60%)
* Somatic mutation
* Unilateral: in one eye
* Unifocal: Single tumor in the eye
* After treatment:
No further risk to develop a new retinoblastoma or other body tumors
The “two hit” model by Alfred G. Knudsen (1971)
FAMILIAL RETINOBLASTOMA
1) mutant Rb allele
2) first somatic mutation
3) two mutant Rb gene copies
-> bilateral disease
SPORADIC RETINOBLASTOMA
1) first somatic mutation
2) mutant Rb allele
3) second somatic mutation
4) two mutant Rb gene copies
-> unilateral disease
° Inheritance of one defective Rb gene (first hit)
° Only one successive genetic alteration in one of the retinal cells (second hit)
°Inheritance of the two “wild-type” Rb gene
°Tumor formation requires two successive genetic alterations in a retinal cell
> both Rb copies must be affected before the disease is manifested
Types of mutations in the Rb1 gene
Mutation types in the first mutated Rb1 gene
➢ nonsense mutations: code for a stop codon that can truncate the protein
➢ missense mutations: code for a different amino acid
➢ frameshift mutations: change of the reading frame resulting in a completely
different translation from the original
➢ small deletions/insertions (indels)
➢ splice site mutations (the Rb1 gene contains 27 exons)
➢ large deletions
Mutation of the second Rb1 gene
➢ loss of heterozygosity (most frequent)
➢ CpG methylation in the promoter region
Loss of heterozygosity (LOH)
MUTANT RB ALLELE
° Heterozygous configuration (Rb+/-): 1 wild type & 1 defective gene copy
° Appearance of wild-type phenotype
TWO MUTANT RB GENE COPIES
° Loss of heterozygosity (Rb-/-): 2 defective gene copies
° Appearance of retinoblastoma phenotype
» Tumor suppressor genes are recessive!
» Both Rb copies must be affected before an effect is manifested
Mechanisms of LOH
aus Rb-/Rb+ kann werden:
Rb-: loss of an entire chromosome by chromosome non-disjunction during mitosis
Rb-/Rb-: loss of an entire chromosome by chromosome non-disjunction and recombination of the mutant allele
Rb-/Rb-: chromosomal translocation
Rb-/Rb-: deletion of the “wild-type” locus (rare)
