Molecular Oncology Flashcards
1
Q
Definition Tumor
A
- Tumor [DE= Schwellung]
- Tumors originate from neoplastically transformed cells
- Tumors consist of tumor cells, immune cells and other cell types
- Clinically relevant: the space used by the tumor (DE: Raumforderung)
2
Q
Formen von Tumoren
A
- Sarcoma : tumors of the connective tissue
- carcinoma : tumors of the epithelia
- Leukemia : tumors of the hematopoietic system
- Lymphoma : tumors of the hematopoietic system
- Tumors of the central nervous system : glioma, gliosarcoma, and others
- Tumors of the peripheral nervous system: Schwannoma, Neurofibroma
- Melanoma: tumors derived from the pigment cells
3
Q
Aging and effects
A
- During life time the cells of an organism begin to age. This may lead to:
- Mutations of oncogenes and tumor suppressor genes -> frequent causes of cancer.
- Dysfunction of telomeres -> several diseases, including cancer.
- dysfunction of repair -> several diseases, including cancer.
- loss of chromosomes and/or changes on the chromatin level -> several diseases, including cancer.
- The capacity to generate functional progenitors and effector cells is reduced.
- Aging of adNSC/adNPC may lead to the accumulation of damages, which are then inherited to the daughter cells.
- The stem cell niche is important as well (affects cell division; regulation of mitosis; „homing“).
4
Q
Aging and senescence
A
- Aging involves several molecular/physiological changes, including shortening of the telomeres.
- Ultimately, the cells will show “senescence”: Induction of senescence may be triggered by ROS or other effectors from the (micro-) environment.
- In some cases, inactivation of tumor suppressor genes overcomes “senescence”: -> proliferation
- This re-entry in proliferation is associated with telomere stabilization/re-activation of telomerase.
- Senescence may be observed in vivo and in vitro (i.e. cell culture systems).
- Senescence in cell cultures may be induced by different parameters, including e.g. “ROS” (reactive oxygen species), “serum deprivation” or simply “progressive passaging”.
- Senescent cancer cells maintain the senescent state by autocrine mechanisms and are influenced by the microenvironment. Paracrine signaling by immune cells has a cancer promoting effect.
5
Q
Difference between tumor cells and normal cells
A
major differences concern:
(1) indefinite growth and replicative immortality,
(2) deregulation of cellular energetics,
(3) evasion of growth suppressors and apoptosis,
(4) genome instability,
(5) promotion of angiogenesis and inflammation.
6
Q
Driver mutations and founder cells
A
- The primary tumor develops from a founder cell.
- The founder cell displays mutations that led to neoplastic transformations.
- More mutations and epigenetic changes are acquired later on, leading to tumor progression and eventually metastasis.
- The founder cell acquired mutations that led to neoplastic transformations.
- These mutations are referred to as “driver mutations”.
- driver mutations accumulate during early stages of tumorigenesis and provide a benefit to the tumor; they occur in “driver genes”.
- not all mutations in a driver gene are supporting cancer.
- Driver mutations lead to a severe dysfunction of the encoded protein.
- Passenger mutations do not provide a benefit to the tumor per se.
- Abundant founder mutations in colon cancer concern the K-RAS proto-oncogenes, APC and Tp53.
- Application of standard radio-/chemotherapy will induce additional mutations. These mutations may erase tumor cell populations.
- A minority of tumor cells may, however, may benefit of the mutations.
7
Q
Tumor classification
A
- Different classification systems exist and are used in clinical settings.
- The tumor classification by the WHO is most often used.
- Specific classification criteria were defined for each tumor entity.
- The suffix “-blastoma” is used to imply a tumor of primitive, incompletely differentiated (or precursor) cells, i.e. refers to
the fact that the tumor is composed of cells resembling precursor cells. - Classification is based on histopathological features. Parameters used in classification include mitotic rate, the Ki67 index, invasiveness, aneuploidy, vascularization and necrosis
- Genetic and epigenetic studies highlighted specific features of tumor entities and subtype of tumors of the same entity.
- Novel grading systems include data obtained by genome, proteome and transcriptome analyses.
- After a tumor is initiated, several factors support or stimulate growth (promotion). Hormones may act as tumor promoters.
- Benign and malignant tumors show several characteristics: e.g. malignant tumors display a increased growth rates, vascularization, necrosis and ulceration. Individual tumor cells disseminate from the primary malignant tumor and form metastasis in a secondary site.
8
Q
Factors that contribute to carcinogenesis are
A
- age, environment and life style
- Free radicals from environment; ROS („reactive oxygen species”) contribute to the various stages of tumorigenesis
- When mitochondria age, the ROS levels are increased; moreover, DNA damage repair is impaired
- shortening of Telomeres is observed during aging.
- Susceptibility: mutations present in the genome are e.g. inherited.
- tumor initiation: requires compounds which deregulate oncogenes/tumor suppressor genes.
- tumor progression: drives progression of already existing tumor cells.
9
Q
Tumorigenesis
A
- Neoplastic transformation converts a normal cell into a “tumor initiating” cell.
- Mutations occur after the cells has been exposed to carcinogens.
- Carcinogenic agents include certain chemicals (e.g. polycyclic aromatic hydrocarbons), high energy beams (X-ray, UV) but also a
subgroup of RNA and DNA viruses. - Tumor promotion may be exerted by many different agents, including hormones and phorbolesters.
- Tumor progression is often associated with genetic and epigenetic changes;
- chronic inflammation and immune evasion are important parameters
- the production of onco-metabolites and the so-called “metabolic switch” are crucial.
- In some rare cases, tumors may be transmitted between animals of the same species (e.g. Tasmanian devil).
10
Q
Tumorigenesis and the “two hit model”
A
- The development of a tumor is a multi-step process, which involves many distinct mutations.
- Several models suggest, that oncogenic “drivers” need to be established during early tumorigenesis. Later on, the functional inactivation of tumor suppressors occurs. Cell cycle, apoptosis and senescence pathways become deregulated.
- Inflammatory processes and epigenetic regulation of additional genes contribute to tumor development.
- Crucial events are the activation of the oncogenic potential of one or more proto-oncogene(s).
- Additional crucial events are the functional inactivation of one or more tumor-suppressor(s).
- In most cases, both alleles of a given tumor suppressor gene are mutated in cancer cells (by e.g.: point mutations, small or large deletions, translocations, promoter deregulation).
11
Q
The types of “hits”
A
- The mutational “hits” may be LOFs (loss of function) or GOFs (gain of function mutation).
- In addition one entire allele (or chromosome) may be lost, which is referred to as “loss of heterozygosity” (LOH).
- Epigenetic changes may lead to repression of tumor suppressor genes (e.g. promoter hypermethylation). – E.g.: BRCA1 and BRCA2;
- Mutations in promoters may lead to drastically impaired levels of tumor suppressors (e.g. BRAC1).
- BRAC1 is important for maintenance of genomic integrity. BRCA1 mutations contribute to breast and ovarian cancer.
Mutations in the BRCA1 promoter may inactivate the gene. - Epigenetic changes may also lead to de-repression of oncogenes (e.g. promoter de-methylation).
- Oncogenic drivers are acting in signaling cascades (e.g. EGFR/HER1, ErbB2/HER2, PDGFR, RAS, RAF).
12
Q
Oncogenic signaling pathways
A
Several proteins are acting together in signaling cascades (modules). The deregulation of the signaling cascades may happen at any point. However, crucial mutations have been identified by the TCGA initiative (included: genome, transcriptome and proteome analyses - and more). Similar signaling cascades are important for distinct tumors, however, the relative importance of individual factors and the type of mutation differ.
- RTK/MAP: Receptor tyrosine kinase deregulation (RTK overexpression; RTK GOFs), deregulation of RAS (GOF) and RAF (GOF)
- Pi3K/AKT/mTOR: Deregulation of the kinases Pi3K (GOF) and AKT (GOF). Functional loss of the phosphatase PTEN (LOF, LOH)
- Wnt/beta-catenin: Deregulated Wnt signaling involves mutated APC (LOF) and deregulated GSK3b (LOF, GOF) and b-catenin (GOF).
- Cell cycle: Controlled cell cycle progression by E2F is impaired or lost due to (i) overexpression of e.g. CDK4/6 or E2F, (ii) mutant pRB (LOF), and/or (iii) mutations in the CDKN2A locus (LOF of p16-INK4A) or (iv) loss of the CDKN2A locus (LOH).
- Myc transcription factors: Myc transcription factors are downstream of RTKs and Wnt signaling. GOFs of cMyc and N-Myc contribute to cancer initiation and the metabolic switch in tumors.
13
Q
Oncogenic activation
A
- Several cellular genes (proto-oncogenes) may be converted into oncogenes (c-oncs).
- Typically, these genes encode regulators of proliferation, cell survival, evasion of growth control and apoptosis.
- The c-oncs encode mitogen or cytokine receptors (e.g. receptor tyrosine kinase EGFR/HER1, HER2, PDGFR), components of signaling cascades (e.g. RAS, PTEN), kinases in signaling cascades (e.g. RAF, Pi3K, AKT, JAK2) or transcriptions factors (e.g. cMyc).
- The activation of the oncogenic potential of proto-oncogenes can occur by/after:
- retroviral transduction (e.g. c-erbB[=HER1]; v-SRC)
- single point mutations (e.g. HER2, JAK2)
- multiple mutations and/or small deletions (e.g. RAS)
- amplification of gene copy numbers (e.g. EGFR/HER1, PDGFR)
- translocation and fusion to a gene, which alters protein function (e.g. t(9;22) BCR-Abl)
- translocation under the control of a highly active promoter (e.g. t(8;14) immune globulin promoter/cMyc).
- demethylation of promoters (formerly repressed c-onc genes are activated thereafter)
14
Q
Proto-oncogenes - Some effects
A
- overactive HER1/EGFR, HER2 or PDGFR support proliferation and cell survival (via MAPK or Pi3K/AKT/mTor cascades)
- Dysfunctional RAS and RAF affect MAPK signaling.
- BCR-Abl drives proliferation and cell survival (via several oncogenic cascades)
- Overactive cMyc augments proliferation and cell survival, and impacts on metabolic pathways.
15
Q
Tumor suppressors
A
- Several proteins may act as tumor suppressors.
- In general, both alleles encoding a tumor suppressor are lost/inactive in tumor cells.
- Several tumor suppressors regulate cell cycle progression:
- (1) pRB [Retinoblastoma gene product] and p16-INK4a;
- (2) Tp53 and p14-ARF.
- Note: p16-INK4A and p14-ARF are encoded by the CDKN2A locus
- The inactivation of tumor suppressors can occur by/after:
- point mutations (e.g. pRB, Tp53; PTEN)
- mutations and/or small deletions (e.g. pRB, Tp53; PTEN)
- loss of heterozygocity (e.g. pRB, Tp53; CDKN2A locus; PTEN) - hyper-methylation of promoters (e.g. BRCA1; CDKN2A locus) - mutations in the promoter (e.g. BRCA1)
16
Q
Specific features of Tp53
A
- Tp53 is a transcription factor, which binds as a tetramer to responsive elements in Tp53 target genes.
- Target genes encode: (i) MDM2, (ii) the cell cycle inhibitor p21, (iii) repair proteins, and (iv) some pro-apoptotic proteins.
- Frequent Tp53 mutations include LOFs and GOFs. Mono-allelic LOFs may exert a dominant negative effect.
- Tp53 GOFs display new functions and are oncogenic. Tp53 GOFs contribute to proliferation and survival, genomic instability, invasiveness, migration and metastasis, immune evasion, angiogenesis and a so-called “metabolic switch”.
17
Q
Tumor micro-environment (TME)
A
- The TME impacts on tumor initiation. tumor progression, tumor maintenance, tumor recurrence and metastasis
- the TME includes: stromal composition, cell-cell interaction, cell-matrix interactions, abnormal physiology, molecular heterogeneity and cellular heterogeneity.
- Tumor cells interact with “host tissue molecules” and the host immune system impacts on tumor growth and immune evasion. The interaction is bidirectional and referred to as tumor-host interaction.
- Neoplastically transformed cells will only generate a tumor in an abnormal micro-environment. This includes non-tumor cells and an abnormal ECM (extracellular matrix), which becomes disorganized and promotes cellular transformation.
- When a tumor grows, the production of new vessels (blood and/or lymphatic vessels) is necessary. Vessels are important for access to oxygen and nutrients, but also metastasis. Release of VEGF by tumor cells supports (lymph-)angiogenesis.
- Hormonal signaling contributes to tumor growth (e.g. tumor promotion by steroid hormones). Hormone therapy may blocks abnormal hormonal signaling in cancer. However, endocrine signaling pathways and micro-environmental cues may promote hormone-resistance and lead to hormone-resistant, aggressive phenotypes.
- Metabolic alterations in tumor stroma occur early and influence the adjacent tumor cells. Metabolites secreted by tumor and non-tumor cells affect tumor growth and progression. Glucose, Tryptophan and Arginine are crucial components.
- The glycosylation pattern of tumor cells is abnormal. It impacts on malignant transformation, cellular growth and migration.
- Several non-tumor cells are present in the TME. This includes cancer-associated fibroblasts (CAFs), myofibroblasts, endothelial cells, adipocytes, mesenchymal stem cells, and various types of immune cells (e.g. cancer-associated macrophages (MAFs).
- In particular, CAFs and MAFs have a major influence on tumor growth and tumor progression.
18
Q
Adaptions, CAFS and the aging tumor micro-environment (TME)
A
- Cells and other components in the TME may show aged phenotypes and undergo senescence. This impacts on tumor initiation, progression, maintenance and relapse.
- One crucial aspect is the tumor cell plasticity: I.e. tumor cells adapt to changes in the TME and undergo genetic, epigenetic, and phenotypic changes throughout tumorigenesis. TME and cancer therapies may select for tumor cells with rare-preexisting genetic phenotypes. Together, these processes increase intratumoral heterogeneity (ITH).
- Adaptations caused by epigenetic alterations may be counteracted by inhibitors of chromatin modifying enzymes.
- Fibroblasts support cellular and microenvironmental homeostasis via regulated secretion of cytokines, chemokines and other signaling proteins. In young, healthy tissues the fibroblast secretome provides a growth-restrictive micro-environment for premalignant cells.
- Fibroblasts undergo a short-lived senescence-associated secretory phenotype (SASP); SASP fibroblasts are quickly cleared by the body in young tissue. In aged tissue SASP fibroblasts accumulate and establish a tumor-permissive, chronic inflammatory micro-environment.
- In addition, Fibroblasts regulate tissue structure via extracellular matrix (ECM) deposition
- SASP fibroblasts promote extensive ECM remodeling. Thereby, they increase tumorigenesis, invasion of tumor cells and immune cell trafficking.
19
Q
Extracellular Matrix
A
- ECM: Components of the ECM (e.g. integrins) serve as ligands for cell surface receptors and provide extrinsic cues that can alter a cells phenotype. Elastic ECM is observed in brain, lung and breast, stiffer ECM structures are seen in skin and bone.
- ECM integrity and elastic properties decrease when an organism ages; ECM stiffness is increased in aged tissue.
- ECM dysregulation is associated with tumor progression and metastasis in hard and soft tissue. Increased crosslinking and stiffening of the ECM will promote tumor progression.
- Age-induced accumulation of SASP is a key mechanism in induction of ECM stiffness in site-specific niches, which may ultimately allow increased tumorigenesis and tumor cell dissemination.
20
Q
Cancer stem cells
A
- The TME displays a cellular heterogeneity. This includes the presence of various non-cancer cells (CAFs, MAFs, MSCs, adipocytes, endothelial cells etc. – see previous summaries). In addition, clones of cancer cells exist in the TME.
- A major role in tumor progression and relapse is ascribed to cancer cells with similarities to stem cells: these cancer cells express a subset of pluripotency factors and display indefinite self-renewal. Hence they are referred to as cancer stem cells (CSCs.)
- CSCs are neoplastically transformed cells, which make up only a small proportion of the tumor mass, but sustain tumor growth.
- CSCs contain the driver mutations that initiated neoplastic transformation. In addition, the CSCs accumulate further mutations and
epigenetic changes. Hence, CSCs with genetic and epi-genetic differences may be present in the same tumor. - Positive and negative selection by the tumor micro-environment will act on the various CSCs present in a given tumor.
- Only a subfraction of the CSCs is able to disseminate from the primary tumor. These CSCs undergo EMT (epithelial-mesenchymal transition) and enter the blood stream. Having reached a suitable new location the CSCs undergo MET to occupy the metastatic niche. EMT and MET are regulated by factors present in the tumor micro-environment.
- Several models address the origin of CSCs:
- (i) CSCs might be derivatives of somatic stem cells, which accumulated mutations and underwent neoplastic transformation. In this scenario, the cells were already stem cells when neoplastic transformation occurred.
- (ii) CSCs might be derivatives of somatic progenitors, which accumulated mutations and underwent neoplastic transformation. In this case, the cells had to switch on genes that augmented the degree of stemness.
- (iii) CSCs might originate from terminally differentiated, mature cells, which underwent neoplastic transformation and were converted into stem cells. In this case, the cells had to re-activate genes that mediate stemness.
21
Q
micro-RNAs and cancer
A
Synthesis and processing of miRNAs are affected in cancer:
- Micro-RNAs are synthesized as a precursor, the pri-miRNA.
- The pri-miRNA is processed to pre-miRNA by DROSHA and DGCR8, both of which might be mutated in cancer cells.
- The pre-miRNA is exported into the cytoplasm by Exportin 5, which is also mutated in same cancers.
- Further processing of the pre-miRNA in the cytoplasm is by DICER and TRBP.
- Finally the miRNA duplex is converted into the single-stranded mature mi-RNA and loaded in the RISC complex.
- Mutations in the genes encoding DICER, TRBP and the Argonaute protein AGO2, respectively, have been observed in cancer cells.
- Together this observations highlight the importance of miRNA for cancer. miRNAs interfere with normal function of proto-oncogenes and tumor suppressor genes.
- The let-7 miRNA cluster affects the proto-oncogenes c-myc and ras, as well as the genes coding for DICER1 and CDK6.
- Moreover, the let-7 miRNA cluster impacts on stemness, modulating the stemness state of cancer stem cells.
- The mRNAs coding for the transcription factor E2F and the tumor suppressor PTEN are affected by the miRNAs.
- Notably, several miRNAs interfere with the stability of the PTEN mRNA: these include miR.221/222.
miRNAs affect chemo-resistance and are therefore crucial in anti-cancer trials:
- miRNAs which increase chemo-resistance include the miR.221/222.
- miRNAs which decrease chemo-resistance include the let-7 miRNAs.
- Hence, several miRNAs may be useful in anti-cancer therapies, whereas other miRNAs will interfere with a beneficial therapy outcome.
22
Q
Cancer Metabolism
A
- Cancer metabolism is abnormal. This includes carbohydrate, amino acid and lipid metabolism.
- Cancer cells undergo a metabolic switch, which includes several strategies and affects several metabolic pathways.
- On the one hand, the changes represent a metabolic adaptation of the tumor cells to changes in the tumor micro-environment (TME). On the other hand, tumor cell metabolism impacts on the TME (e.g. changes in pH due to release of large amounts of lactate) and promotes genomic instability and epigenetic changes.
- Non-tumor cells in the TME supply metabolites (direct release of metabolites or release in form of exosomes) to the neighboring tumor cells – and vice versa. Also, tumor cells supply metabolites to other tumor cells.
- The metabolic adaptations include bioenergetics [ATP production by glycolysis and/or OXPHOS], biosynthesis [activation of pathways synthesizing or metabolizing carbohydrates, nucleotides, lipids, or amino acids] and adaptations of the redox- balance [NAD(P)+/NAD(P)H, ROS].
23
Q
Metabolism in the tumor micro-environment
A
- One major cause for metabolic adaptation/metabolic switch is the reduced or low oxygen (hypoxia) content in the TME. Only in close proximity of the blood vessels, the concentrations of oxygen and nutrients are high. Hypoxia contributes to therapy resistance, vascularization, metastasis and epithelial-mesenchymal transition (EMT). Moreover, Hypoxia stabilizes the transcription factor HIF1a, which would be hydroxylated and subsequently poly-ubiquitinated and degraded under normoxic conditions. HIF1a is one major player in metabolic adaptation, the other is c-Myc. Both transcription factors co- operate in up-regulation of transporters (GLUT, MCT1,4), but have opposite effects in adjusting mitochondrial activity.
- HIF1a up-regulates glucose uptake, lactate release and most glycolytic enzymes (directly or via up-regulation of miRNAs). At the same time entry of pyruvate in the citric acid cycle (TCA) is prevented and OXPHOS is shut-down. This is mediated by the inhibition of the PDH (pyruvate dehydrogenase) complex with help of the kinase PDK1, which is activated by HIF1a.
24
Q
Aerobic glycolysis and Warburg effect; role of fat
A
- Otto Warburg discovered that cancer cells produce energy predominantly by glycolysis; this happens in hypoxic TME (anaerobic glycolysis), but also in presence of O2 (aerobic glycolysis). The phenomenon is referred to as “Warburg effect”. Warburg linked mitochondrial respiratory defects in cancer cells to aerobic glycolysis. It remains to be clarified, if the Warburg effect plays a causal role in cancers or if it is an epiphenomenon in tumorigenesis.
- In tumor cells, the pyruvate produced by glycolysis is further converted to lactate. This overcomes glycolysis-mediated NAD+ depletion in anaerobic tumor cells. In tumor cells with aerobic glycolysis (lactagenic cancer cells) excessive lactate production is a key element of the metabolic switch (i.e. the dysregulation of glucose and lactate metabolism).
- Lactate may leave the cell or is transported into the mitochondria. Both is mediated by monocarboxylate transporters [mitochondrial MCT (mMCT), or MCT1, MCT4 in the plasma membrane). In the mitochondria, lactate may be converted into pyruvate by the lactate dehydrogenase (LDH). Lactate released into the extracellular space lowers the pH (acidification of the TME) and has an impact on tumorigenesis: lactate (i) supports angiogenesis; (ii) increases cell motility, migration and dissemination; (iii) promotes immune escape, decreases T cell activation and cytotoxic activity (iv) enters by neighboring tumor cells and non-tumor cells (via MCT1, MCT4) .
- Obese patients are at increased cancer risk, which is explained by the presence of dysfunctional adipocytes and pre-adipocytes. These cells release fatty acids (FFAs), adipokines and pro-inflammatory factors. The inflammatory environment and the FFAs promote cancer.
- HIF1a, c-Myc and Tp53 contribute to the metabolic switch in tumor cells: they increase glucose uptake by GLUT1. Moreover, lactate dehydrogenase (LDHA) activity is promoted by HIF1a, c-Myc and Tp53. Tp53 and HIF1a interfere with entry of lactate into the mitochondria and favor the uptake of lactate by neighboring cells.
- The expression of PKM isoforms (PKM1, PKM2) contributes to the metabolic switch: both pyruvate kinases (PK) are encoded by the same gene (PKM gene). When differential splicing includes exon 9 the PKM1 is produced; inclusion of exon 10 generates PKM2. PKM1 functions as tetramer and is always highly active. PKM2 forms an active tetramer or a quite inactive dimer. ROS (reactive oxygen species), tyrosine kinases and several oncogenes (e.g. cMyc) lead to formation of the PKM2 dimer. In the presence of the PKM2 dimer, glycolysis is slowed down and intermediates accumulate. Glucose-6 phosphate and the intermediates enter the pentose phosphate pathway (PPP).
- The PPP produces NADPH and building blocks for biosyntheses, which is very advantageous for tumor cells.
25
Metabolic compartments
- anabolic cancer cells
- catabolic tumor cells
- therapeutic aspects
- Cancer cells communicate and cooperatively regulate their metabolism. In the TME anabolic tumor cells reside next to catabolic ones.
- Anabolic cancer cells metabolize glucose by the glycolytic pathway, transport pyruvate into the mitochondria and further metabolize it in
the citric acid cycle (TCA). The reduction equivalents generated in TCA are used in OXPHOS to drive ATP synthesis. Moreover, anabolic cancer cell take up lactate and free fatty acids (FFAs) from the extracellular space and use these metabolites to drive the TCA and OXPHOS.
- Catabolic tumor cells carry out aerobic glycolysis and release catabolites, such as lactate. TCA and OXPHOS are largely inactive.
- Whether a cancer cells is an anabolic or a catabolic cells depends on the neighborhood and the distance to the vasculature: anabolic cancer cells are close to the vasculature and have access to nutrients and oxygen. Moreover, catabolites, such as lactate or ketones, are taken up by anabolic cancer cells. Ultimately, these metabolites enter TCA and OXPHOS. Mitochondrial activity is high; ATP production is efficient .
- Therapeutic aspects: Since the metabolic adaptation of tumor cells supports tumor progression, invasiveness and metastasis, it appears conceivable to interfere with the various aspects of the metabolic switch and/or exchange of metabolites/catabolites. One option is inhibition of transporters (GLUT, MCTs). In addition, agents such as metformin and arsenic disrupt crucial pathways and are expected to result in disturbance in cancer cells energy balance.
26
Metabolic switch and stemness
- Cancer stem cells, like normal stem cells, prevent accumulation of cytotoxic and harmful metabolites, such as reactive oxygen species (ROS). A major source for ROS are the OXPHOS complexes I, III and IV. Hence, reduction of OXPHOS will drastically reduce the amount of harmful and mutagenic ROS. This is not needed in proliferating progenitors and differentiating cancer cells. CSCs, however, prevent accumulation or production ROS by several ways:
- First, quiescent stem cells and CSCs maintain their mitochondria at an immature level (small mass, few cristae, few enzymes and few functional OXPHOS complexes).
- Second, CSCs block further degradation of pyruvate in the TCA by inhibiting the entry reaction catalyzed by the pyruvate dehydrogenase complex (PDC). This is mediated by the PDC-specific kinase PDK, which is e.g. activated by HIF1a.
- Third, CSCs produce antioxidants, such as glutathione (reduced form = GSH) which counteract ROS. The presence of GSH depends on the presence of NADPH, which may be produced by the pentose-P pathway (PPP), IDHs (NADP-dependent isocitrate dehydrogenases) and ME1 (malic enzyme 1). Mutant IDHs, however, consume NADPH in the production of the onco-metabolite 2-hydroxyglutarate (2-HG).
27
Hematological neoplasms
are cancers of the blood, bone marrow, lymph nodes
28
Leukemia
is a cancer of the cells in blood or/and bone marrow; the term covers a broad spectrum of diseases; typical: abnormal increase of blood cells, usually leukocytes
29
Lymphoma
is a cancer of the lymphocytes; may develop in the lymph nodes, spleen, bone marrow, other organs or blood; -> eventually they form a tumor; two subgroups were defined:
1. Hodgkin’s lymphoma (more radiation sensitive)
2. non-Hodgkin’s lymphoma (less radiation sensitive)
30
Myeloma (=MM, myeloma, plasma cell myeloma, Kahler's disease)
cancer of the plasma cells which are derived from bone marrow and are transported through the lymphatic system; the plasma cells (plasma B cells, effector B Cells) produce antibodies.
31
Acute lymphoblastic leukemia (ALL)
- childhood cancer (age: 1 and 7 years)
- cancer of immature lymphocyte cells (=lymphoblasts)
- an ALL subtype shows the Philadelphia chromosome (Ph)
32
Acute myeloid leukemia (AML)
cancer of the immature myeloid cells
33
Chronic lymphocytic leukemia (CLL)
- cancer of the lymphocyte cells.
| - most common type of leukemia in adults (rare in children)
34
Chronic myeloid leukemia (CML)
- cancer of the neutrophils cells.
- a CML subtype shows the Philadelphia chromosome (Ph)
- rare in children, affects adults.
35
Symptoms leukemia patients
feeling sick, fever, chills, night sweat; flu-like symptoms; feeling fatigued; nausea feeling of fullness (enlarged liver, spleen); When leukemic cells invade the central nervous system neurological symptoms (e.g. headaches) occur; due to malfunctioning of immune system cutaneous manifestations are observed.
-> QUITE UNSPECIFIC
36
Symptoms Non-Hodgkins' Lymphoma patients
Swollen lymph nodes; unexplained weight loss; fever; night sweats; coughing, trouble breathing or chest pain; weakness and tiredness; pain; swelling or a feeling of fullness in the abdomen.
-> QUITE UNSPECIFIC
Both, Hodgkin and non-Hodgkin lymphoma may be related to infection with EBV (Epstein Barr Virus)
37
Classification and Nomenclature (acute vs chronic; lymphoblastic vs myeloid)
Classification criteria are (i) acute versus chronic, (ii) lymphoblastic cancer versus myeloid cancer.
Acute versus chronic:
(a) acute: rapid occurrence and progression; rapid accumulation of immature blood cells; malignant cells spill over into the bloodstream and spread to the organs; most common form of leukemia in children.
(b) chronic: progress takes months or years; increase of relatively mature, but abnormal, white blood cells; cancer cells are produced at a much higher rate than normal cells; treatment immediately but sometimes only monitoring for long time periods before treatment starts; at any age, but mostly in older people.
lymphoblastic versus myeloid cancer:
(a) Lymphoblastic (lymphocytic): cancerous change in the marrow cell that normally differentiates into lymphocytes, affects B cells, T cells and NK cells.
(b) change in the marrow cells that normally differentiate into white blood cells; Myeloid (myelogenous): cancerous affects dendritic cells, granulocytes and macrophages, red blood cells, platelets.
38
Neoplasm - Mantle cell lymphoma (MCL)
- Mantle Cell lymphoma (MLC): aggressive, rare, form of non-Hodgkin lymphoma (NHL) that arises from cells originating in the “mantle zone” of secondary lymphoid organs, where B cells mature (hyper-mutation, DNA rearrangements).
- Two major driver mutations have been identified: (i) inactivation of the kinase ATM; (ii) t(11,14) translocation: the CCND1 gene is inserted into the heavy chain (IGH) locus on chromosome 14, leading to over-production of Cyclin D1 (in 90 % of MCL cases).
39
Neoplasm - Burkitt's lymphoma (BL)
Three variants exust: (i) the classical African or endemic type of Burkitt’s lymphoma; (ii) the sporadic type (non-African type), and (iii) the immunodeficiency-associated variant (HIV-infected patients; immunosuppressed transplanted patients).
- chromosomal changes are often observed in patients with Burkitt’s lymphoma; highly frequent: t(8;14)(q24;q32).
- 8q24: c-myc proto-oncogene.- 14q32: immunoglobulin heavy chain gene locus (IgH).
- the t(8;14) translocation brings the c-myc gene under the control of the IgH promotor, leading to c-Myc over-expression.
40
Neoplasm - chronic myeloproliferative disorders
- Mutations of receptor tyrosine kinases (e.g. cKit, PDGF receptors) and Janus kinases, JAKs, (most often JAK2).
- JAKs show a common domain structure: receptor interacting domain (JH3-5, JH6-7), pseudo kinase domain (JH2), kinase domain (JH1). JH2 regulates the activity of the kinase domain JH1 and thereby exerts an “auto-inhibitory effect”.
- Two JAKs bind to a cytokine receptor dimer; the kinase of the one JAK phosphorylates the second JAK; moreover, the JAKs phosphorylate the STAT signaling molecules.
- Replacement of valine 617 by phenylalanine in the JH2 domain (=JAK2 V617F) abrogates the inhibitory effect of JAK pseudokinase domain onto the kinase activity. This leads to a partially active conformation.
- The JAK2 V617F may trigger neoplastic transformation of myeloid cells ( -> chronic myoproliferative disorders).
41
C-Myc dysregulation
- Proper function of c-Myc is needed at all levels of differentiation of B-lymphocytes
- The translocations t(8;14); t(2;8); t(8;22) bring the c-myc proto-oncogene under the control of an immunoglobulin
promoter: this induces overexpression of c-myc and contributes to Burkitt’s lymphoma, multiple myeloma and other cancers. Additional activation mechanisms leading to neoplasms of the hematopoietic system include (i) increased mRNA stability, (ii) epigenetic deregulation of the promoter, (iii) c-Myc self-induction and (iv) increased c-Myc protein stability.
42
cABL dysregulation
- cABL is a tyrosine kinase which is encoded by the proto-oncogene c-abl (human chromosome 9).
- cABL is present in the cytoplasm and the nucleus; it regulates proliferation & survival, and affects the cytoskeleton.
- The t(9;22) (= Philadelphia chromosome; Ph) translocation may generate three distinct BCR-ABL fusion proteins due to variations in the BCR moiety. The Ph is sufficient to induce leukemia and, in the respective contexts, necessary to maintain leukemia.
- The wildtype BCR (= breakpoint cluster region) protein: is a Ser/Thr kinase; is a GEF for Rho GTPases
- All BCR-ABL fusion proteins display oncogenic functions; i.e. they interfere with DNA repair and they possess a constitutively active ABL tyrosine kinase. Moreover, BCR provides the ABL tyrosine kinase with a oligomerization domain and BCR directs the fusion protein to the cytoplasm.
- Many signaling pathways are affected by BCR-ABL: cell adhesion and migration is altered; DNA repair is disrupted, proliferation and survival is increase, BCR-ABL is “anti-apoptotic”.
- cABL and BCR-ABL are inhibited by Imatinib. In leukemia, Imatinib may interfere with function of deregulated cKit, PDGF receptors alpha and beta.
43
Acute promyelocytic leukemia (APL)
- > Molecular analyses identified specific translocations in APL (acute promyelocytic leukemia)
- > APL patients show increased numbers of promyelocytes due to a differentiation block.
- > Dysregulation of RARa (retinoic acid receptor alpha) signaling after translocation is critical.
- > The t(15;17) translocation fuses a truncated RARa to a truncated PML (Promyelocytic leukemia protein)
44
RAR alpha (retinoid acid receptor alpha)
Retinoic acid (RA) regulates development and homoeostasis and plays a role in cancer. RA binds to and activates RAR receptors; RAR/RXR heterodimers recognize and bind DNA, i.e. so-called RAREs;
45
PML
- PML is a nuclear protein and a tumor suppressor
- PML is part of a complex nuclear structure the so-called PML bodies.
- PML regulates several pathways PML activates several proteins and inhibits the function of others.
46
PML-RARalpha fusion protein
- in the fusion protein, the PML N-terminus is replacing the RARa N-terminus
- PML-RARa homo-oligomerizes, binds to RAREs and heterodimerizes with RXR on promotors/enhancers.
- PML-RARa oligomers replace wild-type RARa on RAREs; this results in stable repression of RAR target genes.
- RAR target genes include genes which are essential for hematopoietic differentiation.
- On such promoters/enhancers RARa interacts with PU.1.
- PU.1 is a transcription factor essential for hematopoietic differentiation;
- PU.1 regulates differentiation or activation of macrophages or B- cells.
- PML-RARa homo-oligomers recruit HDACs and prevent activation by Pu.1 -> differentiation block.
- Relieve of the differentiation block is possible by treatment with all-trans retinoic acid (atRA) or arsenic.
- atRA will dissociate the PML-RARa oligomer and allow for access of wild-type RARa to the promotor.
- Arsenic permits sumoylation of PML-RAR and subsequent proteasomal degradation.
47
The hematopoietic stem cells (HSC)
- resides in a niche in the bone marrow; a quiescent (dormant) and a dividing HSC co-exist in the niche;
- The HSC generates a common multipotent progenitor which is then specified for the lymphoid or the myeloid lineage. - The common lymphoid progenitor generates B, T and NK cells
- The common myeloid progenitor generates red blood cells, platelets, macrophages, granulocytes etc.
- Self-renewing HSC can be isolated from the bone marrow
- HSCs generate multiple lineages
- HSCs are able to reconstitute a functional hematopoietic system after transplantation
- HSCs are able to maintain a functional hematopoietic system after transplantation
48
The leukemia stem cells (LSC)
- LSCs reside in a niche in the bone marrow
- LSCs have acquired genetic and epigenetic modifications
- LSCs are derived from HSCs or hematopoietic progenitors after neoplastic transformation
- Transformation involves mutations, translocations or deletions
- LSCs can be isolated from the patients
- LSCs are capable of long term self renewal
- LSCs generate all cells of the neoplasm
- Immature B cells rearrange their genome and show somatic hyper-mutation;
- If these changes do not occur properly, they might be the cause for neoplastic transformation.
49
Cell replacement therapy
is not the first choice! Radio-/chemotherapy will be tried first! Leukemia involving c-ABL fusion proteins, as well as PDGFR or cKit deregulation may be treated with Imatinib, an inhibitor of tyrosine kinases with a kinase insert.
50
Stem cells for cell replacement therapy can be gathered from different sources
- The best source: matched sibling donor
- The bone marrow is a reservoir of distinct stem/progenitor cells and fibroblasts. The stem and progenitor cells include: hematopoietic stem (HSC) and progenitor cells (HPC), as well as mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC). Aspiration of bone marrow from the hip crest is performed.
- In addition, umbilical cord cells (stem cells obtained from umbilical cord blood) from healthy siblings may be used.
51
Treatment most often consists of a 3-day chemotherapy and a total body irradiation (TBI)
-> After chemotherapy and TBI, the stem cells are applied by blood transfusion
-> REQUIREMENTS
- Risk: the transplanted cells have to functionally integrate in the HSC niches (= homing).
- HSCs are mobilized from the donors bone marrow. Mobilization and homing are mirror process depending on an interplay between chemokines, chemokine receptors, intracellular signaling, adhesion molecules and proteases.
- The interaction between SDF-1/CXCL12 and its receptor CXCR4 is critical to retain HSCs within the bone marrow.
- G-CSF disrupts the CXCR4/SDF1a retention axis and, hence, stimulates the mobilization of hematopoietic progenitors cells (HPC) from bone marrow.
- Additional key molecules that regulate HSC function and retention in the bone marrow are VCAM-1 and kit ligand SCF.
52
DNA Tumor virus: EBV
- EBV (Epstein Barr Virus) -> e.g. Burkitt‘s lymphoma
- HPV (human Papilloma virus) -> e.g. oral and cervix carcinoma
- HBV (Hepatitis-B Virus -> chronic hepatitis) -> hepatocellular carcinoma - Beta-Herpes simplex (CMV) -> various tumors, including malignat glioma
EBV infection my lead to transformation of B cells -> this may cause Burkitt's lymphoma
53
Two types of EBV proteins
- Epstein-Barr nuclear antigens: EBNA1, EBNA2, EBNA3, and EBNA-LP
- Latent membrane proteins LMP1 and LMP2
54
Early stages of transformation and later stages of EBV infection
- EBNA2 drives cellular proliferation through c-MYC.
- EBNA2 cooperates with EBNA-LP for LMP1 and LMP2 expression. LMPs are involved in transformation
- Induction of apoptosis by p16INK4a and BIM is blocked by EBNA1A and EBNA3C.
Later stages:
- LMP1 and LMP2 activate NFkB transcription and lead to complete B cell transformation.
55
DNA Tumor virus - HPV
- Viral infection per se does not lead to cancer.
- viruses lyse the permissive cells.
- In the non-permissive state (= viral genome persists in
the infected cell), however, cancer may be induced.
-> Viral Tumorigenesis is a "by-product" (complication) of DNA virus replication and virus-transmission.
56
HPV - Molecular basis
- Viruses exert molecular parasitism.
- Certain viral proteins contribute to/mediate neoplastic transformation. This may e.g. occur by interfering with the function of tumor suppressors, such as p53 and pRB.
- Viruses shift cells from G0 phase into the cell cycle.
57
HPV proteins in neoplastic transformation
- E7 induces a hyper-proliferation;
- E6 blocks apoptosis;
- E5 contributes to the immortalization of cells. E1-protein is a target protein of effector caspases 3 and 7
- E1 is a replication protein
58
The HPV E7 protein interferes with
- Genomic stability
- normal proliferation
- normal apoptosis
59
The HPV protein E6 leads to
- Telomerase becomes more active: higher vitality and immortalization
- p53 is inhibited: Abnormal proliferation, less cell death, higher mitotic rate.
- pro-apoptotic factors such as Caspase 8 and Bax are affected: reduced apoptosis.
60
RNA-Tumor virus: HTLV-1
- HTLV1 targets mature CD4+ helper T-cells -> HTLV-1 induces certain types T cell leukemia in adults.
- Infection is via the mucosa; Cell-cell transmission occurs via the so-called the “virological synapse”.
61
HTLV-1 genome
- LTR: long terminal repeats
- pol: virus polymerase (RT)
- gag: group-specific antigens
- env: envelope proteins
- Important HTLV-1-specific genes encode Tax, HBZ and REX.
62
HBZ
- Basic leucine zipper factor
- Inhibits cellular transcription factors.
- interferes with function of co-activators CBP and p300.
63
REX
- export of the unspliced and singly spliced viral RNAs species from the nucleus.
64
Tax: the "Onco-protein"
- transactivator of viral RNA transcription
- modulates function of cellular factors
Tax is the major contributor to neoplastic transformation
- The Tax protein activates two survival pathways. This promotes survival and proliferation of HTLV-1 infected cells. These pathways are (i) NFkB and (ii) Pi3K/Akt.
- The Tax protein causes multipolar mitosis through the amplification of centrosomes during interphase. This interferes with regular mitosis and results in production of aneuploid cells.
- The Tax protein interferes with the function of the tumor suppressor p53, the repair protein Ku80 and the DNA polymerase b. The repair functions are grossly disturbed, including NER (nucleotide excision repair), BER (base excision repair) and MMR (mismatch repair): -> this leads to increased chromosomal abnormalities.
65
Animal models decipher (epi-)genetic pathways in glioma
- The genetic and epi-genetic aberrations leading to malignant glioma were established in mouse models. In addition the tumorigenicity of GBM cells and their responsiveness to treatment were analyzed in mouse models. As an alternative, zebrafish might be used.
- Xeno-transplantation of human glioblastoma (GBM) cells into immunosuppressed mouse models. Disadvantage: human cells are analyzed in a mouse environment. Preferentially SCID mice are used, as their severe combined immunodeficiency affects B and T lymphocytes.
- Heterotopic model: the GBM cells are xeno-transplanted subcutaneously: GBM growth is analyzed outside the brain.
- Orthotopic model: the GBM cells are xeno-transplanted into the mouse brain: GBM growth is analyzed in its “natural” environment.
- As an alternative, these assay may be performed in zebrafish embryos. Advantage: GBM cells could be observed in the living animal.
- GEM [genetically engineered] (autochthonous) models: these are genetically modified mouse models. The mutations (or epigenetic changes) suspected to contribute to GBM development are introduced into the mouse genome. The brain (and other organs) of the mouse are observed for GBM development. Advantage: influence of the tumor micro-environment and non-tumor cells can be monitored.
- In combination with data collected with human GBM biopsies the GEM models identified two major GBM types, the GCIMP and the non- GCIMP. In G-CIMP tumors aberrant methylation and/or histone mutations contribute to a high extent to the tumor phenotype. Mutations concern the histone H3K27 (Lysyl 27) mutant and epigenetic silencing of e.g. IDH1 expression. In addition, loss of DNA methylation at specific loci causes disruption of CTCF binding sites, which results in reorganization of chromatin and dysregulation of gene expression.
- In non-GCIMP glioblastoma: four GBM subtypes were proposed, which are referred to as the (i) classical/proliferative GBM, (ii) the mesenchymal GBM, (iii) the neural GBM and (iv) the pro-neural GBM. Specific mutations and LOH are associated with the four GBM subtypes, which occur in temporally defined sequences.
- The pro-neural GBM (PN) displays many similarities to secondary GBMs.
- Crucial mutations in primary GBMs (except for the PN subtype) are the overexpression and/or deregulation of EGFR, as well as Tp53 and PTEN mutation or loss. Crucial mutations in secondary GBMs are IDH1 inactivation, loss or mutation and the mutation of ATRX. Childhood
GBMs are rare: Crucial mutations in pediatric GBMs are the deregulation of RAF and the mutation of ATRX.
66
the TCGA data
- The TCGA initiative analyzed the genome, transcriptome and proteome of tumor biopsies from three selected tumors (breast cancer, pancreatic cancer and glioblastoma multiforme [GBM]). Later on the analyses were extended to other frequent and infrequent tumor entities. Moreover, methylation pattern, microRNAs, metabolic aspects and factors involved in angiogenesis and invasiveness were investigated. The TCGA initiative provided additional evidence for the four GBM subtypes: the (i) classical/proliferative GBM (CL), (ii) the mesenchymal GBM (Mes; M), (iii) the neural GBM (N) and (iv) the pro-neural GBM (PN).
- The following crucial mutations were assigned:
- CL GBM: EGFR (mutation, amplification), TP53 mutation
- Mes GBM: NF1 mutation, TNF and NFkB deregulation; NF1 is a regulator of RAS activity and, hence, affects MAPK cascade signaling.
- N GBM: expression of neural proteins, deregulation of EGFR signaling
- PN GBM: IDH1 mutation, PDGFRa amplification, abnormal PI3K signaling.
67
the GBM micro-environment
- Angiogenesis: Effectors from the GBM micro-environment impact on angiogenesis. Hypoxia and HIF1a are the major inducers. angiogenic factors (VEGF, FGF) are released and several proteases (e.g. MMPs) are produced and activated.
- Migration and invasiveness: the extracellular matrix (ECM) is degraded by MMPs, which are produced in the GBM microenvironment. Integrins activate the FAK (focal adhesion kinase) and in turn several down-stream effectors, including Rac1. Rac-1 is activated by stress
fiber responses and/or the PI3K/AKT pathway. Rac-1 is important for development of protrusions needed for migration.
- Proven risk factors: (i) environmental: irradiation; (ii) hereditary syndromes associated with high glioma risk affect e.g. Tp53, NF1, IDH1/2.
- Glioma cells (and GSCs) cross-talk to non-GBM cells in the tumor microenvironment (TME): (i) GSCs secrete VEGF, which in turn contributes to angiogenesis; (ii) GSCs respond to growth factors provided by the TME and neighboring cells; (iii) the GSCs secrete factors that influence the non-GSCs in the TME. The most important cross-talks are with (A) endothelial cells and (B) microglia and TAMs.
- Microglia and TAMs (tumor-associated macrophages) secrete TGFb, which in turn stimulates the secretion of proteases (e.g. MMPs) by GBM cells. The GBM cells produce versican, which activates the MMPs and induces the secretion of proteases (MMPs) by TAMs.
- GAMs (= microglia + TAMs) are recruited to the tumor lesion by glioma cell-derived factors.
68
Melanoma incidence
Definition: Melanoma is an aggressive fatal form of skin cancer originating from the melanocyte (neoplastically transformed melanocyte). Early stages of melanoma can be surgically removed with little risk of recurrence. Metastatic melanoma, however, is associated with high rates of mortality.
Incidence: Melanoma is related to life style. During life, 1 out of 60 persons will develop invasive melanoma, 1 out of 32 non-invasive melanoma in situ. CSD (chronic-sun damage), non-CSD, acral, mucosal and uveal melanoma exist. Acral melanoma forms on palms, soles and in the nail bed. Mucosal melanoma may develop on all kinds of mucosa (e.g. gingiva, intestinal and genital mucosa).
Melanin: Melanin protects skin against UVA and UVB; melanin is synthesized by the melanocyte and delivered to the keratinocyte in form of melanosomes. In addition, melanin is transferred between keratinocytes. Melanocytes possess filopodia that are necessary for UVR-stimulated melanosome transfer; several factors control melanin synthesis; a key factor is the aMSH receptor (regulates e.g. mammalian hair and skin color).
Melanin synthesis starts with the amino acid Tyrosine, goes via Dopaquinone and produces brown and black Eumelanin.
69
Melanoma risk
Risk factors related to life style -> (i) intermittent UV radiation concomitant with sunburns (both, UVA and UVB are harmful); (ii) fair skin is more sensitive than dark skin; (iii) sunburn with blistering; (iv) previous non-melanoma skin cancer (i.e. basal cell carcinoma, squamous cell carcinoma), (v) large number of moles, (vI) abnormal moles.
Genetic risk factors: the GWA (genome-wide association) studies identified genetic risk factors: these include the gene encoding the aMSH receptor and the CDKN2A locus
70
Benign nevus
sharply-circumscribed lesion derived from melanocytes; brown or blue color;
May be already present at birth or occurs as chronic lesion during life; always composed of melanocytes
71
Dysplastic nevus
larger than common moles; irregular and poorly defined borders; color variations in the nevus (pink -> dark brown); considered pre-cancerous; highly frequent (1 out of 10 persons in US); may develop into cancer, but many of these nevi do not do so.
72
Radial growth phase (RGP)
becomes uneven and more and more asymmetric; areas of partial regression are observed; still confined to the epidermis; referred to as: superficial spreading melanoma and melanoma in situ. Cured by surgical removal. Cells from RGP generate melanomas in mouse models.
73
Vertical growth phase (VGP)
acquires a rich vascular network; local invasion and ulceration; patients show relapse and poor survival rates; the cells from VGP express laminin receptor which enables them to adhere to vascular walls -> first steps in direction of dissemination.
74
histopathology
all melanomas are derived from melanocytes; a subgroup of melanoma cells does not synthesize melanin, this is seen in amelanotic melanoma; Amelanotic melanoma is distinct from basal cell and squamous cell carcinoma; the latter ones do not originate from melanocytes or their prescursors but from epithelial cells.
75
Melanoma progression
Clarks model: Melanoma development is defined by Clark’s model for melanoma progression; the model describes a series of histological changes beginning with the melanocytic nevus. B-Raf mutation (B-RAF V600E) may act as the driver mutation. The observation of that B-RAF wildtype and B-Raf mutant cells are present in the same lesion, suggested that distinct/additional mutations might act in tumor initiation .
the progression stages are:
(i) Benign nevus -> dysplastic nevus
(ii) radial growth phase -> induction of angiogenesis/vasculogenesis
(iii) vertical growth phase -> continued growth and vascularization
(v) metastasis.
76
Genetic stages and associated mutations/epigenetic changes
1. nevus: may carry the B-RAF V600E mutation
2. Dysplastic nevus: disruption of pRB pathway by either (i) mutation in the p16INK4A gene within the CDKN2A locus or (ii) amplification of CDK4-gene.
3. Radial growth phase: immortalization of melanocytes and activation of the telomerase (TERT); another hit in the CDKN2A locus may have occurred.
4. Vertical growth phase: loss of functional PTEN, activation of RAS (proto-)oncogene; in addition, cell surface proteins are affected, i.e. activation of b-Catenin and aberrant Cadherin expression.
Progressive stage 3 -> 4: activation of AKT/PKB may convert radially growing into vertically growing melanomas; continued growth and angiogenesis, since VEGF and pro-angiogenic factors are released.
5. Metastasis: continued angiogenesis and dissemination of melanoma cells
77
Metastasis and dormancy
- when the vertical growth phase is reached, metastasis will occur; the disseminating melanoma cells will spread to other parts of the body and potentially form the secondary tumor.
- The melanoma cells may, however, enter dormancy [i.e. the secondary tumors form >10 or 20 years later];
- in 5 – 10 % of the patients with metastatic melanoma the primary tumor has not been recognized.
- Reasons for dormancy or its relieve include: (i) tumor microenvironment, (ii) proliferation signals, (iii)
composition of nutrients, and (iv) the immune system.
78
Metastatic process and factors involved
- after the tumor has acquired a vascular network, the cells disseminate; this involves progression and the angiogenic switch, which is followed by the switch to the metastatic phase.
- the melanoma cells secrete VEGF and bFGF to attract endothelial cells;
- bFGF promotes growth and attraction of endothelial cells and mast cell invasion;
- dissemination requires the disruption of cellular bonds [i.e.: between the receptor CXCR4 and its ligand
SDF1; between cadherins].
- During the process of dissemination the melanoma cells secrete MMPs; the balance between MMPs and
TIMPs (inhibitors of MMPs) is critical; Expression of the laminin receptor enables the melanoma cells to adhere to the vascular walls.
79
Melanoma - deregulated cell cycle
Melanoma risk is increased in cancer families (i.e. the HBOC and Lynch families)
The cell cycle is deregulated in melanoma. Frequently, this deregulation involves mutations and epigenetic changes affecting the MAP kinase pathway.
80
Melanoma - deregulated proliferation
Proliferation is increased and deregulated, because the MAP kinase cascade is affected at 3 stages:
(i) over-expression or amplification of cKit;
(ii) mutation of RAS or B-RAF (mutual exclusive = either RAS or B-RAF).
(iii) In addition, ERK (is a kinase in MAPK signaling) may be activated by the focal adhesion kinase FAK, which responds to ECM (extracellular matrix)/ Integrin-signaling.
The receptor tyrosine kinase cKit is a regulator of melanocyte migration, survival & differentiation. SCF is the cKit ligand. SCF binding activates ckit.
The activity of cKit may be inhibited by Imatinib.
81
deregulated factors - B-RAF
- the Ser/Thr kinase B-RAF is one of three RAF proteins; however, a single point mutation will only activate the oncogenic potential of B-RAF, but not of A–RAF or C-RAF.
- The oncogenic mutation B-RAF[V600E] is observed in most melanomas (80-90%).
- All RAF proteins possess (i) a RAS-binding domain (RBD), which binds selectively to RAS-GTP (=activated RAS), (ii) a kinase domain and (iii) an auto-inhibitory domain (CR1).
- The B-RAF[V600E] mutant shows increased kinase activity, because the interaction between the auto-inhibitor domain (CR1) and the kinase domain in abrogated.
82
deregulated factors - NER
Any mutation inactivating NER enzymes is crucial, as the NER pathway removes the UV-induced photodimers. TFIIH is a RNAP II transcription factor; defects in the TFIIH subunits XPB (helicase ) and XPD (helicase) impair NER. Hence XPB and XPD mutations induce xeroderma pigmentosum (XP) and contribute to melanoma.
83
deregulated factors - CDKN2A and CDK4
CDKN2A locus mutations are referred to as “high-penetrance melanoma susceptibility locus/gene”. CDK4 is referred to as rare high-penetrance melanoma susceptibility gene.
Both types affect cell cycle progression
- The cell cycle is deregulated in melanoma
- Two major changes are observed: Mutations in the CDKN2A locus and CDK4 de-regulation.
- the CDKN2A locus encodes p16-INK4A and p14-ARF.
- P16-INK4A is a regulator of RB signaling, p14-ARF of Tp53 signaling.
- When p16 is affected then the control of the Retinoblastoma gene product pRB disrupted and cell cycle progression is possible (deregulation of the senescence pathway).
- The mutation of p14 affects p53 function (deregulation of the apoptosis pathway).
- Mutation of p53 itself is rare in melanoma.
- In addition the activation of the telomerase TERT is observed.
84
melanoma-associated factors alphaMSH-receptor (=MC1R)
- G-protein coupled receptor; encoded by MC1R gene and signals via cAMP.
- MC1R binds to a class of pituitary peptide hormones, the melanocortins; this includes the a-melanocyte stimulating hormone. MC1R agonists (α-MSH) increase genomic stability in melanocytes, antagonist (ASIP) decrease it.
- MC1R gene is polymorphic -> major factor in determining skin pigmentation, variants of MC1R appear associated with melanoma risk (-> familial melanoma).
- DNA damage provokes increased production of MSH and MC1R; The activated MC1R induces the expression of MITF via a G-protein cascade. The factor MITF is produced and translocates into the nucleus, where it up-regulates genes involved in pigment synthesis
85
melanoma-associated factors MITF (micropthqthalmia-associated transcription factor; bHLH family transcription factor)
- mutated human MITF in patients with Waardenburg Syndrome IIa (white forelock, deafness ...).
- MITF is a key player in melanocytic development. MITF is activated by c-Kit and aMSH [and b-catenin] signaling cascades (involves modification of protein leading to MITF activation).
- MITF dictates the pigment cell phenotype by regulating melanocyte-specific proteins.
- MITF regulates genes involved in melanoblast survival, lineage commitment, and melanocyte. proliferation and survival; may function as an oncogene in the context of melanoma.
86
Animal models for melanoma
- The analyses of deregulated signaling cascades were established in mice.
- Orthotopic model: tumor cells are xeno-transplanted and orthotopics tumors develop.
- A genetically engineered mouse model proved the role of B-RAF V600E.
Very important models are teleost 3 distinct species of fish:
- Zebrafish -> screening for mutations and epigenetic changes; imaging of melanoma development (from the melanocyte
to the melanoma cell)
- Medaka -> search for oncogenes and tumor suppressors involved in melanoma
- Xiphophorus -> a model for genetic predisposition (the fish carries a melanoma-gene)
-> Spontaneous melanoma (genetically generated hybrids)
-> Melanoma after treatment with carcinogens or X-ray (tumor initiation)
-> Melanoma after treatment with tumor promoters (promotion with hormones)
87
Tumors of the nervous system
Gliomas, Meningiomas
88
Primary Brain Tumors
Neuroepithelial Tumors (gliomas)
- Astrocytomas
- Oligodendrogliomas
- mixed gliomas
- ependymomas
Others:
- Medulloblastomas
- Glioneuronal tumors
Meningeal Tumors:
- Meningioma
Germ cell tumors Lymphomas (PCNSL)
89
Secondary Brain Tumors
Metastasis
- Kidney
- Lung
- Gut
- Skin
- Breast
- Others
90
Intra-axial neuro-epithelial tumors
Astrozytomas, Oligodendrogliomas, Glioblastomas
91
Incidence Brain Tumors
- Gliomas, especially glioblastoma 6/100.000
- Meningiomas 6/100.000
- Pituitary tumors 1/100.000
- Nur(in)Omas (Schwannomas) 1/100.000
- Metastases 10/100.000
92
Gliomas - WHO Classification
```
1 = Pilocytic Astrocytoma
2 = Low-grade (diffuse) Astrocytoma
3 = Anaplastic Astrocytoma
4 = Glioblastoma
```
93
General symptoms
- elevated intracranial pressure (ICP) (Headache, Nausea, Vomiting, Papilledema)
- seizures
- changes of consciousness, mental changes
94
Forms of herniation
- subfalcine herniation
- uncale herniation
- central, transtentorial herniation
- tonsillar herniation
95
Local symptoms
```
Hemiparesis -> frontal, parietal
Hemianopsia -> temporal, occipital
psycho syndrome -> frontal
vision, perimetry -> suprasellar
cranial nerves -> skull base
imbalance of hormones -> sella
```
96
Diagnostik bei Hirntumoren
- Bildgebung inklusive Kontrastmittel, vorzugsweise Kernspintomographie
- Liquoruntersuchung bei Tumoren, die häufig im Liquorraum metastasieren (Medulloblastom, ZNS-Lymphom) Cave! Bei intrakranieller Raumforderung
- (Teil)Resektion oder stereotaktische Biopsie zur histologischen Sicherung
97
Pilocytic Astrocytoms (WHO 1)
Pilocytic astrocytomas, also known as juvenile pilocytic astrocytomas, are circumscribed astrocytic gliomas that tend to occur in young patients. They are considered WHO grade 1 tumors in the current WHO classification of CNS tumors and correspondingly have a relatively good prognosis.
These tumors have a range of imaging appearances, with the majority presenting as a large cystic lesion with a brightly enhancing mural nodule. Calcification can be present in around one-fifth of cases. The majority of pilocytic astrocytomas arise from the cerebellum.
The term pilocytic refers to the elongated hair-like projections from the neoplastic cells. The presence of eosinophilic Rosenthal fibers is a characteristic feature and hyalinization of blood vessels is also common.
Immunohistochemistry reflects astrocytic differentiation:
GFAP: positive, S100: positive, OLIG2: positive, IDH R132H mutation: negative, p53 protein: negative or weak
Pilocytic astrocytoma frequently have BRAF alterations (present in ~70% of cases). Importantly they, along with other pediatric low-grade gliomas, lack IDH mutations and TP53 mutations.
Surgical resection, if complete, is usually curative.
98
Low-grade Glioma (WHO Grade 2)
Low-grade gliomas (LGGs) are a diverse group of primary brain tumors that often arise in young, otherwise healthy patients and generally have an indolent course with longer-term survival in comparison with high-grade gliomas.
Treatment options include observation, surgery, radiation, chemotherapy, or a combined approach, and management is individualized based on tumor location, histology, molecular profile, and patient characteristics. Moreover, in this type of brain tumor with a relatively good prognosis and prolonged survival, the potential benefits of treatment must be carefully weighed against potential treatment- related risks.
99
Glioblastoma (WHO 4)
Glioblastoma, the most common and lethal primary malignant brain tumor in adults, frequently recurs because of tumor heterogeneity, rapid proliferation, and infiltrative lesions.
Initial treatment in patients with glioblastoma — maximal resection followed by radiotherapy and temozolomide — is associated with 14.6-month median overall survival (OS).
Randomized phase 3 studies have shown no improvement in OS after dose-dense temozolomide, or with the vascular endothelial growth factor inhibitor bevacizumab plus temozolomide and radiotherapy.
Adding tumor-treating fields to temozolomide resulted in a 4.9-month survival benefit. Nitrosoureas or bevacizumab provide median survival of 6 to 9 months for recurrent disease.
100
Glioma Therapy
Principles: Lowering of ICP, Tumorvolume reduction, histological diagnosis, regain of function
Biopsy (stereotactic, neuronavigation)
Microsurgery (endoscopy, neuronavigation, CUSA, intraoperative fluorescence)
Irradiation (Radiosurgery, fractionated, interstitial)
Chemotherapy (systemic, local, Immunotherapy, Gene-therapy)
COMBINATION
101
Principles of surgery and typical approaches
- preservation of function more important than radicality
- microsurgery
- bipolar coagulation
- watertight dural closure
- laser is out
102
Chemotherapy of gliomas
Standard: Nitroso-urea: BCNU, CCNU, temozolomid
Problems:
- intratumoral heterogeneity
- blood-brain-barrier
103
MGMT Status
Das O6-Methylguanin-DNS-Methyltransferase (MGMT)-Gen codiert für ein gleichnamiges DNS- Reparaturprotein, welches Alkylgruppen von der Position O6 des Guanins der DNS entfernt. Die Wirkung einiger Chemotherapeutika (wie z.B. Temozolomid) beruht auf Anfügen von Alkylgruppen an diese Position O6 - Zytotoxität und Apoptose der Tumorzellen sind die Folge. Eine erhöhte MGMT- Expression und somit erhöhte DNS-Reparaturaktivität könnte demnach der Wirkung alkylierender Chemotherapeutika entgegenwirken. Die MGMT-Proteinexpression wird über den Promotor des MGMT- Gens reguliert. Eine epigenetische Stilllegung des MGMT-Gens durch Promoter-Hypermethylierung gilt
als Hauptursache reduzierter MGMT-Proteinexpression und somit verringerter DNS-Reparaturaktivität.
104
Isocitrathydrogenase (IDH)
Punktmutationen im IDH1-Gen (Arg132) und seltener im IDH2-Gen (Arg172) treten bei >70% der primären astrozytären und oligodendroglialen Tumore sowie sekundären Glioblastomen (GBM) auf. Bei primären GBMs sind diese Mutationen dagegen in nur ~5% der Fälle zu finden. Sowohl bei GBMs als auch bei anaplastischen Astrozytomen sind diese Punktmutationen mit einer besseren Prognose korreliert
105
1p/19q-Status
Bis zu 2/3 aller oligodendroglialen Tumore zeigen einen kombinierten Verlust der Allele (loss of heterozygosity, LOH) auf dem kurzen Arm von Chromosom 1 (1p) und dem langen Arm von Chromosom 19 (19q), welcher auf eine unbalanzierte Translokation zurückzuführen sein soll. Anaplastische Oligodendrogliome bei denen solch ein kombinierter 1p/19q-Verlust vorliegt sprechen besser auf eine Chemotherapie an. In mehreren retrospektiven Studien sowie zwei prospektiven randomisierten Phase III Studien konnte darüber hinaus der kombinierte 1p/19q-Verlust als unabhängiger Marker für eine bessere Ansprechbarkeit auf Radio- und Chemotherapie sowie eine verlängerte Überlebenszeit bei diesen Patienten identifiziert werden.
106
Glioblastom - Therapie der Wahl
- maximale Resektion (radiologisch komplett)
- kombinierte Radio-Chemotherapie (fraktionierte Bestrahlung und orale Therapie mit Temozolomid (ACNU, BCNU, (Avastin), etc.)
- Tumor treating Fields (TTF)
Temodalgabe ist abhängig von molekulargenetischen Markern, insbesondere dem MGMT-Status.
107
Immunotherapy of gliomas
Problems:
• intratumoral heterogeneity
• immunosuppressive glioma microenvironment
• tumor’s effects on systemic immune function
• stimulation and prolongation of tumor-specific cytotoxic T-
cell activity
• T-cell populations are often transformed to an exhausted
phenotype
Suppressed immunoresponse due to surgery and corticosteroids
108
Possible causes for brain tumors
- no viral factor known
- earlier head or brain irradiation enhances the risk for a glioma at a factor 3-7
- the risk for a meningioma at a factor 10
- no certain association with mobile phone use
- very rare association with genetic syndromes (Li-Fraumeni-, Turcot- Syndrom)
