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)
Germaine mutations are often found in TSGs
Why are mutant tumor suppressor genes transmitted through the germline while oncogenes are usually not?
Oncogene
1. Dominant at the cellular level
- activation of one allele is sufficient to create the neoplastic phenotype, e.g. uncontrolled proliferation
2. Disruption of normal tissue development in the embryo
» An example: Mouse embryos carrying an activated K-ras oncogene die to the time of mid-pregnancy because of placental & embryonic developmental defects
Tumor suppressor gene
1. Recessive at the cellular level
- inactivation of one allele is not sufficient to create the neoplastic phenotype
2. Their presence in most cells of an embryo will not be apparent (no phenotype); normal embryonic development
One rare exception: Ret oncogene
- The oncogene Ret can cause hereditary thyroid carcinoma
- The proto-oncogene Ret encodes a receptor tyrosine kinase
- Constitutive activation of RET promotes unregulated proliferation (oncogene function)
- Thyroid carcinoma
Why can mutated Ret be transmitted through the germline?
* Expression of Ret is limited to a small set of tissues
* Delayed expression of Ret during embryogenesis
» Normal development of the embryo
RET signaling regulates growth and survival of cells
-> Biological function: development of the kidneys and the enteric system
RET: rearranged during transfusion (transfection of human lymphoma DNA into 3T3 fibroblast cells)
Human tumor suppressor protein p53 at a glance
- Protein 53 (p53), 393 amino acids
- Transcription factor
- Stress senor, inhibition of tumor development
- Hindering proliferation of damaged cells (tumor suppressor function)
- Prevent genome mutations (guardian of the genome)
Cellular stress: DNA damage, Lack of nucleotides, activated oncogenes, hypoxia, blockage of transcription
Cellular responses: DNA repair, Cell-cycle arrest, senescence, apoptosis
p53 protein domain structure
Transactivation domains ( TAD1 and TAD2): Transcriptional activation of p53 target genes
Proline-rich domain (PRD): Structural function and protein-protein interaction
DNA-binding domain: Sequence-specific binding to p53 response elements in the promoter regions
Tetramerization domain (Tet): p53 binds to its DNA response elements as tetramer
Note: only the p53 tetramer can efficiently bind to the DNA helix
Lysine-rich domain (Basic) (regulator domain): Non-sequence-specific DNA binding (modulates the sequence-specific binding)
Transactivation of target genes by tetrameric p53
p21 + 14-3-3sigma + GADD45 -> cell cycle arrest/Senescence
p53 R2 + p48 + GADD45 + XPC -> DNA repair
DR5 + Pig3 + AIP1 + Noxa + Bax + Puma + Fâs -> Apoptosis
Mdm2 controls the p53 level through TAD occlusion and degradative ubiquitination
Acidic: Phosphorylation site, regulation of Mdm2 function
RING: E3 ligase activity
Mdm2: murine double minute 2
NLS: nuclear localization signal
Control p53 level through negative feedback loop
- Formation of p53 tetramers (active transcription factor)
- Transactivation of various genes including the mdm2 gene
- Production of Mdm2 protein
- Mdm2 binds p53 in the nucleus (TAD occlusion, inactivate p53)
- Export to the cytoplasm & Mdm2- mediated ubiquitination of p53
- p53 degradation
» Short half-life (~20 min) results in low steady-state level of p53
Mdm2: E3 ubiquitin ligase (murine double minute 2)
p53 stabilization and activation
Activation of DNA-damage sensing kinases leads to p53 stabilization and activation
(1) DNA damage-induced activation of Chk2/ATM and ATR kinases
(2) TAD phosphorylation
- Activation & stabilization of p53 - No binding to Mdm2
- Half-life: several hours
(3) Transactivation of p53 target genes
(4) Inactivation of Mdm2 by multisite phosphorylation and degradation (molecular mechanisms not clear)
Dependent on the stress signal: » Cell cycle arrest & DNA repair or apoptosis (e.g. increased BAX expression)
CHK2: cell cycle checkpoint kinase 2
ATM: Ataxia Telangiectasia mutated gene 7
ATR: AT and RAD related
Nature of p53 mutations
Missense mutations
- lead to amino acid substitutions
- rarely lead to truncated versions
9% = frameshift
75% = missende
2% = in frame deletions/insertions
7% = nonsense
5% = silent
2% = splice site
p53 mutations
The six most common p53 amino acid residues altered in cancer
- Contact mutations: prevent direct DNA binding
- Structural mutations: impair proper folding of the DNA binding domain
Dominant-negative mutation of p53 gene
- p53 normally functions as a homotetrameric transcription factor
- Missense mutation in the DNA binding domain results in the expression of altered proteins which can form tetramers but cannot bind to DNA
- All tetramers with one or more mutant p53 subunits are defective in their function as transcription factor and are more stable than wild type p53 (Knudson didn’t know about p53! Monoallelic mutation!)
Mutant p53 is more stable than wild-type p53
Wild-type p53:
* Mdm2-mediated negative feedback loop results in low steady-state level of p53
* No detection by immuno-histochemistry (see normal, unstressed epithelial cells)
Mutant p53:
* No Mdm2 expression
* No p53 degradation
* Accumulation of p53 protein
* Detection by immuno-histochemistry (see dysplasia and carcinoma, black nuclei instead of blue (DAPI))
Accumulation of mutant p53 in tissue of ovarian carcinoma
Loss of p53 function
- Mutation of one allele
- Change the conformation of wild type p53 (tetramer)
- Dominant-negative mutation
- Inhibition of DNA binding
- Mutation of one allele & LOH of the wild type TP53 gene
- Inhibition of DNA binding
- abnormal increased amplification of MDM2 (oncogene)
- Ubiquitin-mediated degradation of p53
Paradigms of tumor suppression
- Two-hit paradigm: Loss of one allele induces cancer susceptibility; loss of two alleles induces cancer
- Haploinsufficieny: a single copy of wild-type allele at a locus in heterozygous combination with a
mutated allele ➜ although wild-type allele still produces the normal product, the total product is
sufficient for induction of cancer - Quasi-sufficiency: tumor suppression is impaired after subtle downregulation of expression without loss of even one allele
- Obligate haploinsufficiency: TSG haploinsufficiency is more tumorigenic than complete loss of the TSG, usually due to the activation of fail-safe mechanisms following complete loss of TSG expression
APC - Adenomatous polyposis coli
APC is a tumor suppressor protein
1. Inactivating mutations in hereditary and sporadic colon cancers
2. Multiple biological (normal) roles: proliferation, differentiation, apoptosis, adhesion, cell migration, development
APC is a gatekeeper protein
1. Loss of APC function leads to the appearance of unregulated dividing cells
2. Transition from normal epithelial cells to hyperplastic cells
Tumor suppressor protein APC
Adenoma-to-carcinoma progression
- Inactivation of tumor suppressor genes (TGS): loss of APC, loss of 18q TSG (SMAD4), loss of p53
- Activation of oncogenes: activation of K-ras
Domain structures and functions of APC
OLIGOMERIZATION: Homodimers
ARMADILLO REPEATS: protein interaction and cytoskeleton function
BETA CATENIN BINDING: 15 aa repeats
BETA CATENIN BINDING AND DOWN REGULATION: 20 aa repeats
AXIN BINDING: SAMP repeats (axin binding)
-> binding of beta-catenin promotes beta-catenin degradation -> inhibition of transcription, Myc (TF) and cyclin D1 (G1->S) -> Control of proliferation (down regulated) and differentiation (up regulating)
MICROTUBULE BINDING: Kinetochore functions -> Chromosomal segregation
Axin: scaffold protein in the β-catenin destruction complex, which holds the protein complex together.
SAMP (serine-alanine-methionine-proline)
TF: transcription factor
APC is component of Wnt/Frizzled signaling pw
Wnt ligands
-> large family of secreted ligands (19 human Wnt genes)
Frizzled receptors
-> large family of 7-transmembrane-spanning receptors (serpentine) (10 human frizzled genes)
- The Wnt signaling pathway is critical for the regulation of crypt stem cell renewal and tissue homeostasis
- Loss of both APC alleles can lead to the growth of adenoma.
LRP: transmembrane protein (low density lipoprotein receptor-related protein)
Lgr: Leucine-rich repeat-containing G-protein coupled receptor 5
Wnt signaling pathways
Frizzled receptor: OFF
inactive Dsh
beta-catenin destruction complex (Half-life of beta-catenin: <20 min): Amin, APC, active GSK-3beta, beta-catenin
beta-catenin + Ubiquitin ligase (E1, E2, E3)
Proteasome -> Oligopeptides, amino acids
Wnt ligand + Frizzled receptor: ON
inactive GSK-3beta + active Dsh + APC + beta-catenin
Half-life: 2 hours
beta catenin + Tcf/LEF
Transcriptional activation of Wnt target genes (e.g. cell cycle activation of Wnt target genes (e.g. cell cycle activator cycle D1 and Myc)
Increased proliferation
Dsh (mammals: DVL): dishevelled protein (cytosolic phosphoprotein which is activated);
Axin: scaffold protein
GSK-3ß: glycogen synthase kinase 3ß (phosphorylates ß-catenin);
Tcf/LEF: transcription factors
APC mutations
Distribution of germline and somatic mutations found in the APC gene in colorectal tumors
- oligomerization
- armadillo repeats
- beta catenin binding
- beta catenin binding and down regulation
- axin binding
- mutation cluster region
- microtubule binding
Nature of APC mutations
Frameshift and nonsense mutations:
- Formation of truncated APC proteins
- Loss of beta-catenin binding site
- uncontrolled proliferation
frameshift: 51 %
missense: 4 %
in frame deletions/insertions: none
nonsense: 32 %
silent: 9 %
splice site: 4 %
APC in tumorigenesis
An example of a C-terminally truncated APC protein and its role in tumorigenesis
LOSS OF THE BETA-CATENIN BINDING DOMAIN -> Activation of beta-catenin/TCF transcription -> Activation of proliferation and inhibition of differentiation
LOSS OF THE MICROTUBULE BINDING DOMAIN -> induction of spindle dysfunction -> induction of chromosomal instability
Complete absence of APC appears to be disadvantageous
- many truncated APC retain some ability to bind beta-catenin (15 aa repeats) (beta-catenin degradation, regulation of beta-catenin level in the tumor cell?)
-> Aim: growth advantage for the tumor cell
beta-catenin and the pathology of colon crypts
Stems cells
-> Receiving Wnt signals (red arrows) from stromal cells (red).
-> Activation of gene expression through the ß-catenin/Tcf/Lef complex
-> WNT signaling
(i) results in proliferation (ii) prevents differentiation
Progenitors
-> Migrate upward toward the lumen (left side of crypt).
-> Decrease of Wnt signaling
-> Near the lumen, the proliferation rate decreases and the cells start to differentiate
-> Apoptosis after 4 to 6 days (small green arrows)
Defective APC protein (initiation) (right side of crypt)
-> High ß-catenin level without Wnt signaling
-> The mutant cells (APC-/-)
- constitutively proliferate
- do not further differentiate
- do not migrate to the top of the crypt protecting the cells against apoptosis on the top
-> Conversion of normal to hyperplastic epithelium (blue-purple)
-> These mutant cells & their descendents can sustain additional mutations which can promote tumor progression
