Molecular Biology Flashcards
(92 cards)
1
Q
What is cancer?
A
- uncontrolled growth
- breakdown in normal mechanisms
- a disease of ‘self’ cells so harder to incite immune response
2
Q
What are the hallmarks of cancer?
A
- avoiding immune destruction
- enabling replicative immortality
- tumor-promoting inflammation
- activating invasion and metastasis
- inducing angiogenesis
- genome instability + mutation
- resisting death
- evading growth supporessors
3
Q
How does smoking affect your risk of cancer?
A
- tobacco smoke has >80 carcinogens
- 72% of cancer cases linked to smoking
- those who smoke 15-24 a day have a 26x increased risk of cancer compared to never
- risk is 5x for those who smoke 1-5 a day
4
Q
What are the occupational hazards that can cause cancer?
A
- asbestos - lung and mesothelial
- radiation/UV - skin
- radiation/nuclear - leukaemia
5
Q
Which 3 cancers are associated with alcohol intake?
A
bowel, breast, oesophagus
6
Q
Which 2 cancers can be caused by viruses?
A
- cervical (HPV)
- liver (hepatitis)
7
Q
What are the reproductive choices that can impact cancer incidence?
A
menstrual age, birth control choice, having children or not
8
Q
What are the 4 progressive stages of colorectal cancers?
A
- normal colonic epithelium
(APC) - small adenoma (polyps)
(RAS) - large adenoma
(PI3K) - carcinoma
9
Q
What are the 5 progressive stages of breast cancers?
A
- normal duct
- ductal hyperplasia
- atypical hyperplasia
- ductal carcinoma
- DCIS microinvasion
10
Q
What are this differences between benigh and malignant tumors?
A
- benign tumor cells resemble normal cells, malignant less differentiated
- benign tumors dont metastasise
- benign tumor proliferate slowly
- benign tumors don’t need to be treated
11
Q
What are viral-transformed 3T3 cells?
A
- a model system for studying cell transformation, tumorigenesis, and oncogenic signaling
- derived from moouse embryonic tissue, exposed to oncogenic viruses
12
Q
What is luminal A BC?
A
ER+ PR+ HER2- (treated with hormone therapy)
13
Q
What is luminal B BC?
A
ER+ PR± HER2± (treated with hormone therapy + chemo)
14
Q
What is HER2 enriched BC?
A
ER- PR- HER2+ (treated with HER2 targeted therapy)
15
Q
What is triple negative BC?
A
ER- PR- HER2- (treated with chemo)
16
Q
What are proto-oncogenes?
A
- normal function to control cell growth
- converted to oncogenes by ‘gain of function’ mutation
- point mutation always active - gene amplification - more protein - chromosomal translocation
- a gene which encodes a protein able to transform cells
17
Q
What are tumor-suppressor genes?
A
- genes that restrain cell growth, promote cell death and promoyte DNA repair
- loss of function leads to excessive growth
- recessive gene, both copies must be lost
- can be inherited causing familial cancer
18
Q
What is the SRC oncogene?
A
- a proto-oncogene that encodes non-receptor tyrosine kinase
- first found in Rous Sarcoma Virus
19
Q
What does cellular-SRC do?
A
c-SRC is used for
- cell growth and proliferation
- cell adhesion and migration
- angiogenesis
- anti-apoptotic signalling
20
Q
What is the only human oncogenic retrovirus?
A
Human T cell lymphotrophic virus
21
Q
How is src intramolecularly regulated?
A
- src has three major domains, SH2, SH3 and a kinase domain
- src switches from inactive to active through phosphorylation
22
Q
What are viral fossils in the genome?
A
- the human genome contains 8% viral genes
- may host immunity and block viral infections
- recognise specific invades, launch immediate attack against entire classes of virus
- when retroviruses invade they turn RNS in DNA which can become part of genome
- sometime infect gametes and so are passed down to next gen
- endogenous retroviruses cannot produce new viruses
23
Q
What is human papilloma virus?
A
- non-enveloped circular dsDNA, form papillomaviridae family
- 100+ types can integrate into host genome
- results in epithelial lesions and cancers, primarily cutaneous + mucosal surfaces
24
Q
What does HPV do?
A
- HPV infects basal stem cells of mucosa epithelium, viral DNA remains independent prior to integration into host genome at sites prone to breakage
- virus inactive in early infection but keeps cell from entering G0 stage to dysregulate cell cycle
25
What is EGFR?
- acts as a viral entry co-receptor, drives cell proliferation
- a receptor kinase RTK, activation leads tp down-stream activation of RAS-MAPK and PI1K-PKB pathways
26
What are the RTK family members?
- Dysregulation of RTK associated with many diseases
- Genes encoding EGFR, HER2, and MET
4 principle mechanisms
- Gain of function
- Genomic amplification
- Chromosomal rearrangement
- Autocrine activation
27
What are tyrosine kinase inhibitor strategies?
ATP-competitive inhibitors - compete with ATP for binding to the kinase domain, blocking phosphorylation activity (imatinib - BCR- SBL, getfitin - EGFR)
Allosteric inhibitors
mAbs - bind extracellular domain of RTKs, blocking ligand binding or inducing receptor degradation (Cetuximab - EGFR, Trastuzumab - HER2 )
Dual/multi-kinase inhibitors - Target multiple kinases simultaneously to overcome resistance or target several pathways (Sorafenib, Sunitinib - VEGFR, PDGFR)
28
What are the therapeutic challenges for TKI?
- Drug resistance - primary + acquired
- Off-target toxicity
- Limited penetration
- Focus initially on greater specificity to reduce side effects
- Broader acting agents proved more useful (dirtier)
29
What were the first generations of TKIs?
1st gefitinib + erlotinib
2nd afatinib + dacomitinib
3rd osimertinib + olumtinib
30
Targeting HER2/neu
- A receptor tyrosine part of EGFR family - protein kinase erβ-2 resides at the plasma membrane, encoded by ERBB2 gene
- ERBB originally isolated from an avian genome
- Human protein is HER2 or cluster of differentiation
- Located on chromosome 17
Targeted by:
- mAbs - trastuzumab (bind extracellular domain, block dimerization or promote immune killing)
- TKIs - lapatinib (Inhibit intracellular kinase activity)
- ADCs - trastuzumab emtansine (deliver cytotoxic drugs directly to HER2-positive cells)
31
Detection of HER2/neu
- Immunohistochemistry (biopsy tissue staining with monoclonal antibodies under standard conditions), scored 0-3+
- Fluorescent in situ hybridisation (detects gene expression, more sensitive than IHC)
- Chromogenic In Situ Hybridisation (same as FISH but easier to see)
- Enzyme linked immunosorbant assay (ELISA - extracellular domain, detects cleavage products in serum)
32
Gene expression profiling
- Efforts focused in improving therapeutic approach with greater understanding of gene activity driving cancer
Done using technologies like:
- Microarrays: DNA probes on a chip detect RNA transcripts by hybridization.
- RNA sequencing (RNA-Seq): Sequencing all RNA molecules in the sample for detailed quantification.
- Metastatic disease varies from primary cancer cell, main focus
33
Herceptin (trastuzumab)
- A mAb designed to target HER2
- HER2 produced by ERBB2 gene and forms clusters in cell membranes in tumors
- Amplification occurs in 15-30% of BC
Blocks HER2 signaling
- This blocks the downstream signaling pathways (like PI3K/AKT and MAPK) that promote cancer cell growth and survival.
Induces Antibody-Dependent Cellular Cytotoxicity (ADCC)
- Flags the HER2 +ve cells for immune destruction
Promotes receptor internalization and degradation and inhibits angiogenesis
-
- Treats early stage ovarian, uterine, gastric, salivary
- Amplification of HER2 correlates with better survival in esophageal adenocarcinoma + gastric/cardiac
- Potential cardiac toxicity
34
Future directions
- Antibody-drug conjugates use herceptin to target a toxin
- HER2+ disease uses the CNS as a sanctuary
- ~50% of patients develop brain metastasis
- The blood-tumor barrier (BTB) develops from the BBB regulated drug distribution
35
RAS
- Activating mutations (at 12-61) present in many human tumors: 90% pancreatic, 45% colorectal
36
RAS as anticancer?
- Fatty acid modification which tethers it to membrane
- Inhibitors of this developed peptidomimetics
- Good response in mouse breast tumors model
- Clinical trials - disappointing, MoA unclear
- K-Ras appears active w/o extras
- Potential therapeutic target in various forms of leukaemia
37
Cell cycle regulation
- Checkpoints at different points to check each stage of replication
- Many therapies utilise high energy usuage of cancer cells and starve them but this takes away energy from normal energy dependent cells
- Cells remain in G0 until induced to replicate decreased checkpoints
38
Tumor suppressor genes
Cell cycle regulation - stops cell division when conditions aren’t right
DNA repair - fixes mutations before cells divide
Apoptosis promotion - triggers cell death in damaged or abnormal cells
Inhibiting metastasis - prevents spread of cells to other parts of the body
39
germ cell cancers
- Cancers can form in somatic + germ cells, both are controlled by multiple proteins that govern DNA replication and cell cycle advancement
- Germ cells tumors can appear at any age, developing from cells that produce gametes, sacrococcygeal, brain, chest, abdomen
40
somatic regulation vs germ line regulation
- Different properties in mitotic/meiotic properties
- Lots of TSGs indentified and these tend to congregate
- Good starting point for treatments as many common themes
- Somatic mutations not inherited
41
Defective TSGs
- The proto-oncogene + TSG work together to control cell cycle
- Both must be engaged for normal cell function
42
Hereditary disposition
- Cancers tend to have inactivated mutations in one or more TSGs, inherited germ line mutations in one allele and subsequent somatic mutation in the other
- Deletions/point mutuations result in no protein or protein with altered function
- Defects in mismatch repair + excision genes predispose to cancer
- Wnt and hedgehog function in development with loss of patched 1 function, enabling cells in absense of Hh
- Defects in APC drives precancerous intestinal polyps with increased risk of colon cancer
- BRCA1 defects result in increased (60%) probability of BC compared to 2% with two WT alleles
- Hh tells cells to stop growing even with excess GF as no more room
- Once defective, waits for proto-oncogene and then cancer
43
Targeting BRCA
- BRCA genes protect cells by controlling pair of DNA repair system
- Cells that proliferate rapidly wont stop and check
- Issues in DNA very likely, probable cause of mutation
- Some mistakes kill cells, other cause evolution
- Cell has more drive to metastasise
- Without BRCA 1/2 proto-oncogene can escape
- When gene detected, possible phylactic surgeries removing breasts, ovaries, and cervixes
44
Retinoblastoma
- Tumors develop in the retina, 40% of cases inherited
- Retinal tumors early in life, one or both eyes
- If recessive, only need proto-oncogene, no TSG
45
Retinoblastoma protein regulation
- LOF pRB make E2F TF constitutively, making cell growth independent of cyclin D levels
- LOF p16 removes cells ability to halt cell cycle to repair DNA damage
- mutations pass to daughter cells and accumulate, 3 relevent mutations needed to transform cell
- HPV E7 subunit pRb
46
Tumor protein 53
- p53 is the most frequently mutated gene in human cancer
- p53 protein is a tetrameric TF known as the guardian of the genome to to its critical function in preserving integrity
- p53 normally present at low levels, complexed to MDM2 that inhibits action
- Stress signals inhibit MDM2, allowing activated p53 to regulate expression of p16,21,27 proteins and Bad/Bax pro-apoptotix proteins
- This allows p53 to cause cell cycle arrest + induction of apoptosis - HPV E6 subunit inhibits p53
47
p53 correction systems
- When extra p53 added to normal cells, became cancerous, as balance key
- p53 can be activated by many carcinogens like cigarette smoke
48
What are the 4 p53 stresses?
DNA damage
Oncogenes
Loss of survival signals
Hypoxia/anoxia
49
What are the 5 p53 responses?
cell cycle arrest
differentiation
DNA repair
apoptosis
senescence
50
p53 as a therapeutic agent
- p53 rarely mutated in cervical cancer but polymorphism affects suspectibilty to HPV E6
- Chemotherapeutic agents + radiation reply on inducing apoptosis for cytotoxic agents
- Lack of p53 often makes tumor cells resistant through lack of functional pathway
- Research focused on restorinh p53 function
- CDB3/PRIMA-1 stabilises mutant p53 function + restores transcriptional function
- SMIs to inhibit MDM2 binding to p53 are entering clinical trials
51
Main points of TSG
- Potential oncogenic events occur constantly as mistakes in replication events occur at non-zero frequency
- Inherant mechanisms of repair/cell clearance are engaged to resolve mistakes
- Most mutations probably lead to non-viable cell outcomes that are never of note, but some lead to gain of function for growth drivers as well as loss of function for repair/cell clearance pathways
- Physiochemical properties of specific genomic sites lead them to have bias toward specific mutations leading to enhanced frequency of specific mutations in the population
- Two-hit or more hypothesis put forward since no single mutation appears significant to site oncogenesis
- Mutations in oncogenes and suppressor genes can be identified in most cancers
52
Approaches to targeted therapies
- Identification of dysregulated molecules/pathways
- Requires genetic profiling of cancer
- Easier to target overexpression/activation than LoF
- Challenges - endogenous molecules/ubiquitous pathways
53
Leukaemia
- Upregulated proliferation of a clone of immature white blood cells
- Squeeze normal cells out of bone marrow
- Blood appears milky
54
Classification of leukaemia
- acute or chronic
- myeloid/lymphoid (based on origin)
55
How does chronic myeloid leukaemia present?
- fatigue, anaemia, splenomegaly, hepatomegaly
- elevated white blood cells
- all stages of granulocytic differentiation on blood smear
- hypercellularity of bone marrow
- increased ratio of myeloid to erythroid cells
- 20% of adult leukaemias
56
What are the 3 clinical phases of chronic myeloid leukaemia?
1. initial milder chronic phase
2. accelerated phase develops after ~ 4 years
3. acute leukaemia phase - blast crisis
57
Mutations in chronic myeloid leukaemia
- Mutations thought to arise in stem or progenitor cells
- 95% of CML has reciprocal translocation between chromosomes 9+22
- Generates fusion between break point cluster region
58
Chromosomal translocation
Moving one gene to another chromosome would make too much difference
Rate of transcription controlled by promoter region
- Some regins undergo high transcription rate others is slower
- High rate = lots of mRNA, lots of protein and vise versa
- If you move a gene from low to high, may result in over expression and vise versa
- If oncogenes move low to high - overexpression
- TSG from high to low, decreased expression
- In leukaemias, only half gene moves, often translocated with another gene
59
C-Abl
- Non-receptor protein tyrosine kinase (Src family)
c-Abl itself isn’t always oncogenic in its native form, but it becomes a potent oncogene when abnormally activated, especially in BCR-ABL
- Normally in cytoplasm but under stress can translocate to nucleus - upregulate gene transcription to protect/prepare cell
- Activated by ionising radiation/DNA damage
Nuclear C-Abl
- Complexes with p53 to induce cell cycle arrest + DNA repair/apoptosis
- Bound by pRb inducing cell cycle arrest
60
BCR-Abl
- Unable to leave cytoplasm, can't bind with cytoplasm (patients known as philadelphia chromosome +ve)
- However, novel protein to target
- SMIs for the kinase (specifically ATP binding site)
61
Imatinib
- SMI, inhibits proliferation of human CM2-derived cell lines, inhibits CML growth in mouse model
- Blocks activity of Abl tyrosine kinase
- Also blocks PDGFbetaR and c-kit tyrosine kinases (also involved in cancer)
62
Imatinib phase I trials
- well tolerated
- 300mg/day, complete haematological response
- cytogenic response - decrease in phy cells
- fast-tracked
63
Next generation inhibitors
- Dasatinib/nilotinib bind active (not closed) so good for resistance
- 3rd gen SAK2 inhibitor (tozasertib) initial promise but cardiotoxic
64
TEL-PGDF beta receptor fusion
- A. genetic abnormality where parts of two different genes become abnormally fused due to a chromosomal translocation,
- Associaoted with chronic myelomonocytic leukaemia
- Translocation between chromosomes 5+12
- Kinase always active, helix + loop, helix region of TEL induces oligmerisation
- Fusion can transform in culture
65
Glivec (imatinib) + GIST
- GI Stromal Tumors
- Driven by constitutive c-kit receptor activity
which is blocked by glivec
66
SMIs
- Often receptor tyrosine kinase inhibitors
- Block triggering pathways
- Orally active
- Specificity issues
67
Monoclonal antibodies
- Only target extracellular molecules
- Highly specific but prone to mutation induced resistance
- Require IV, expensive
68
Pharmacogenomics and personalised therapy
- Pt + tumor variability leads to variation in treatment response
- eg Iressd - more effective in pt carrying mutation leading to hyperactive EGFR
- Pt with mutation in K-ras won't respond to EGFR inhibitors
- Variation in drug metabolising enzymes can lead to chemo toxicity
69
Reconstitution
- More challenging to 'restore' LoF
- IV particle or nanoparticle mediated delivery - advexin
- Targeting is key
- Delivery is challenging
- Alternative is to target over activity of molecule no longer being suppressed
70
Embryonic stem cells
- Pluripotent can differentiation into different cell types
- Unspecialised cells that can reproduce themselves and/or generate more specialised cells in definitely self-renewal
- Intermediate cells known as pre-cursors of progenitor cells
- Resulting specialied often cannot divide - specialised function
- Terminal differentiation generally irreversible
- Change in cell state is transcriptionally + epigenetically regulated
71
Assymetric stem cell division
Stem cell > stem cell (totipotent)
\/ restricted potential SC > RP (multipotent)
\/ progenitor SC > progenitor (unipotent)
\/ terminally differentiated SC
72
Adult stem cells
- Small numbers of residual stem cells in adult tissues
- Include blood, intestine, skin, muscle, liver, brain
- In bone marrow, stem cells for normal cell tumor
- In liver + muscle, stem cells for healing
- Only in very small numbers, proliferation suppressed
- Difficult to identify + isolate
73
Stem cells in cancer
- If undifferentiated, stem cells are implanted into tissues, can form tumors
- Differentiated cells can dedifferentiate and reacquire ability to proliferate
- Alternatively, mutations may occur in tissue stem cells
- These proliferate rapdily, undergo some differentiation and metastasise more easily
74
Stem cells and cancer
- Stem cells live long + self-renew, giving more opportunity for accumulation of mutations
- Assymetric division may account for heterogenity of tumor mass
- Adult stem cells present at low numbers in tissues but can recolonise
- Many signalling pathways involved in self-renewal are mutated in cancer cells
75
Stem cell regulation
- Stem cell proliferation usually regulated by niche stem cells
- Niche cells secrete factors which supress or stimulate stem cell proliferation
- Cancer stem cells can become niche-independent or under control of a different niche
- Pathways controlled by transcription factors that upregulate/supress self-renewal
76
Cancer stem cell hypothesis
- Expansion of normal stem cell niche permits expansion of cancer stem cells that arose from normal cells
- These cancer cells adapt from a different niche allowing expansion
- The cancer cells adapt from a different niche allowing expansion
- The cancers cells become niche-independent and self-renewal is autonomous
77
Wnt and Hedgehog pathways
- Wnt is a proto-oncogene pathway mutated in 90% of colon cancers
- Wnt binding is rec Frizzled sequesters GSK-3 releasing beta-catenin to act as TF to drive c-myc and cyclin D expression and hence proliferation
- Hh - sonic, desert and indian binds to patched (TSG) and releases inhibition of smoothened signalling pathways then release Gli TF to drive proliferation
78
Transit - differentiation of intestinal Villi cells
- Showed mutating APC in crypt cells caused tumors but not in prog or differentiated cells
- Restoring APC function restores APC function, crypt cell function, even in presence of other mutations
79
Targetting Wnt in cancer
- Appropriate site of intervention depends on mutation
- Wnt ligand + receptor inhibitors (porcupine inhibitors decrease Wnt ligand secretion, mAb to Wnt receptor of ligand)
- Axin inhibitors (tankynose (PARPi) stabilise axin)
- b-catenin inhibitors, disrupts TF function to drive self-renewal
80
Telomerases
- Tandem repeats on the ends of chromosomes and aid chromosomal replication
- Because the DNA polymerases cannot copy right to end of the chromosome, telomeres shorter with each cell division
- When the telomeres get too short, they are recognised as damaged DNA and p53 is activated to induce senescence or even apoptosis
- Telomerases are reverse transcriptase enzymes containing RNA template to add TTAGGG repeats
- Telomerases are found rapidly dividing + germ-line cells, most somatic cells lack telomerases to repair chromosomes and extend life
- Telomerase and N-myc levels indicative of prognosis
- Potential target for anticancer therapy
81
Challenges of using stem cells
- Heterogenity of tumor mass
- 3D hypoxic nature of tumor mass
- Potential harm of WT stem cells
- Drug reistance - ATP - binding - cassette (ABC) transporter
82
Angiogenesis
- Growth of new blood vessels
- Blood vessels bring O2 + nutrients to cell removes CO2 + waste
- Vasculogenesis during embryogenesis is programmed
- Angiogenesis involves formation/maturation /differentiation of vessels from pre-existing vessels
- Tumor neoangiogenesis is not regulated or programmed
- Increased angiogenesis correlates with oor prognosis
83
Process of angiogenesis
- Tumor expressed Vascular Endothelial Growth Factor (VEGF), gradient towards tumor
- VEGF binds to specific receptors (VEGFR) on endothelial cells
New vessels have
- Disorganised structure
- Low inter-endothelial cell junction
- Low pericyte coverage
- Increase microvascular permeability (leaky)
- Low integrity vessels that can collapse
- Low perfusion
Can be utilised for chemo?
- Once a tumor gets bigger than 1-2mm3 it needs a blood supply, cells are hypoxic
84
3 Activators of angiogenesis
- VEGF
- PDGF
- bFGF (p53)
85
3 Inhibitors of angiogenesis
- endostatin
- angiostatin
- thrombospondin (p53)
86
The 4 anti-angiogenic therapies
monoclonal antibodies
- anti-VEGFA antibody, bevacizumab
- anti-VEGF2 antibody, ramucirumab
decoy receptors
- aflibercept binds to VEGF-A/B
receptor tyrosine kinase inhibitors
- sorafenib + sunitinib (multi-kinase inhibitors)
others
- thalidomide analogues inihib phosphorylation of AKT, crucial for signalling of GFS
87
Metastasis
- Different cancers use different growth factors
- breast cancer tends to use platelet derived growth factors
- Metastasis is the ability of cancer cells to escape primary tumor via blood + lymphatic to grow in second site
- 90% of cancer mortality related to advanced metastatic disease
- 45% cancer diagnosed at stage 3-4
- always test lymph nodes when diagnosing
88
The metastatic cascade
- Primary tumor growth - proliferation
- Angiogenesis
- Detachment + invasion into surrounding tissue towards vessels
- Intravasation into lymphatic/capillaries
- Survival in circulation
- Arrest in new/secondary organ (small capillaries, adhesion to vessel wall)
- Extravasation into secondary tissue
- Establishment of microenvirnoment (death, dormancy, proliferating)
89
Epithelial-mesenchymal transition
- The majority of life-threatening cancers occur in epithelial tissues
- For motility and invasiveness, carcinoma cella must change phenotype from a more epithelial to a mesenchymal phenotype
Epithelials
- Cytokeratin expression
- Adherance junction
- Epithelial cell polarisation
- Epithelial markers: E-cadherin, beta-catenin
Mesenchymal
- Fibroblast-like shape
- Increased motility + invasiveness
- Secretion of proteases (MMPs)
- Mesenchymal markers: N-cadherin, vimentia
90
Tumor micro-environment
Immune cells
- T cells, T reg, dendritic cells
Macrophages
- M1 pro-inflammatory, anti tumor
- M2 less inflammatory, pro tumor
Cancer-associated fibroblasts
- driving EMT, digesting cellular matrix and letting cell escape
Cancer cells
91
Theory of site specific metastasis
- First pass organ (tumor cells carried through bloodstream and recolonise in next organ encountered)
- Seed + soil hypothesis (provision of a fertile environment supports growth of tumor cells)
- Preparation of pre-metastatic niche by myeloid-derived suppressors and tumor exosomes
- Compatible adhesion molecules on endothelial cells
- Appropriate GF and ECM
- Selective chemotaxis
92
Targetting the metastatic cascade
- MMPIs - 1st gen Marimastat - poor efficacy, high toxicity - lacking specificity - potential for alpha-MMP
- VEGFR/MET (EMT receptor) inhibitor - cabozantinib
- Targeting metastatic recolonisation (MET)
- Dormant disseminated micrometastatic tumor cells (evidence they can be reactivated by fibrosis so testing antifibrotic ab pamreulumab)
- Targeting bone resorption - metastases tobone can reupregulate RANKL to active osteoclasts and cause bone resorption
