Week 7 Flashcards
What are the hallmarks of cancer
Sustaining proliferative signalling- cancer cells stimulate their own growth
Evading growth suppressors- cancer cells resists inhibitory signals that might stop cell growth
Resisting cell death- evade apoptosis
Enabling replicative immortality- cancer cells have a limitless replicative potential
Inducing angiogenesis- stimulation of growth of BV to get better nutrient supply to tumour
Activating invasion and metastasis- spreads cancer to distant sites
Emerging hallmarks
Avoiding immune destruction
Deregulating cellular energetics
Enabling characteristics
Genome instability and mutation
Tumour promoting inflammation
Metabolic transformation is required to permit cancer hallmarks
Self sufficiency in growth signals- proliferation required- new proteins, DNA, RNA and ATP
Insensitivity to anti growth signals
Sustained angiogenesis- new endothelial cell proliferation, survival in hypoxia- glycolytic ATP generation, new proteins, DNA
Tissue invasion and metastasis- cell movement, production of MMPs- new proteins, ATP
Evasion of apoptosis- change in mitochondrial phenotype- alterations in mitochondrial metabolic pathways
Replicative immortality- ability to replicate indefinitely
Nutrients
Lipids- cell membranes, energy
Carbohydrates- DNA, proteins, cell membranes, energy
Proteins- proteins, DNA, energy
ATP- energy currency
Glycolysis- conversion of glucose into pyruvic acid
End products: 2 ATP, 2 NADH, and 2 pyruvate molecules
NADH is fed into electron transport chain where it regenerates itself into NAD+ and produce more ATP
However this reaction requires more oxygen
If O2 is insufficient NAD+ has to be regenerated by fermentation process such as conversion of pyruvate acid into lactate
Fermentation in cancer
Metabolism in cancer cells:
-cancer cells have increased glucose uptake
-they also run glycolysis at a much higher rate to produce ATP and divert glycolytic intermediates to bio synthetic pathways
-very little pyruvate goes into mitochondria for oxidative phosphorylation
-the majority of it is converted into lactate to regenerate the NAD+
In normal cells:
-cell takes up glucose and metabolises majority into pyruvate
-this enters mitochondria where it is metabolised through the TCA cycle
-energy is then produced via oxidative phosphorylation
-only a very little amount of pyruvate is converted into lactate
Energy versus macromolecules
Because cancer cells proliferate so fast there’s always a trade off between energy production and production of macromolecules
Cancer cells divert carbons to macromolecules biosynthesis but by doing this they sacrifice ATP production
This is the reason they run glycolysis at such high rate
The Warburg effect
Describes an increased lactate production by cells under aerobic conditions
Misunderstandings about Warburg:
-Warburg effect can never be observed in hypoxia
-it does not necessarily describe increased aerobic glycolysis, which is not unique to cancer cells
Why is the Warburg effect always talked about
We observe increased lactate production in cancer cells and in most tumours
Its an indicator of metabolic transformation of tumour cells but there are a number of different ways of getting this effect
How does the Warburg effect and other transformed metabolic phenotypes occur
Oncogene/tumour suppressor gene induced changes in proliferative drive and direct modulation of metabolism
PTEN activity lies downstream of many signalling pathways
PTEN: phosphatase and tensin homolog deleted from c10
Negatively regulates the PI3K-AKT pathway
Activation of this signalling pathway in melanoma occurs through loss of PTEN, autocrine and paracrine growth factors and adhesion receptor signalling
If we lose PTEN this pathway can happen and AKT is activated
This supports cell survival, proliferation and invasion
AKT activation increases glucose uptake
Via translocation of GLUT glucose transporters from the cytoplasmic vesicles onto the cell membrane surface
AKT and glycolysis
AKT activates the enzyme phosphofructokinase
This is a pace setter for glycolysis
So AKT makes glycolysis happen faster
P53- a central metabolic regulator
Has several functions that increase oxidative phosphorylation thereby opposing the Warburg effect
-p53 functions include transcription and activation of expression of SCO2 which is a key regulator of cytochrome C oxidase complex
P53 also opposes Warburg by inhibiting glycolysis via
-expression of hexokinase
-inhibition of PGM
-represses GLUT 1 and 4
P53 helps maintain mitochondria and drives oxidative phosphorylation
P53 controls oxidative phosphorylation through SCO2
The reduced dependence on oxidative phosphorylation for energy production shown by cancer cells is not generally due to defect in components of TCA cycle or electron transport chain but reflects an ability of proteins associated with oncogenic transformation to promote glycolysis
These not only include AKT but other oncoproteins associated with deregulated proliferation such as MIC and responses to oxygen starvation
P53 deficient tumours therefore exhibit the Warburg effect
Because of mitochondria deactivation as well as less inhibition of glycolysis
What happens if TP53 is mutated instead of knocked out
Retention of its ability to upregulate enzymes involved in:
-detox oxidative stress
-DNA/RNA synthesis
-oxidative ATP generation
-increased glucose consumption
C-Myc transforms glutamine metabolism
Myc regulates genes involved in lipogenesis, ribosome biogenesis, glycolysis and glutaminolysis
The myc max hetro-dimeric transcription factor is shown bound to an E box along with another anonymous TF (TFI)
Both regulate genes involved in lipogenesis, nucleotide and protein synthesis and myc also regulates genes involved in glucose and glutamine metabolism to provide ATP and anabolic substrates for bio mass accumulation
Myc amplification often observed in tumours-> increases expression of myc target genes
Why is c-Myc amplification so food at driving proliferation
Glutaminolysis provides a source of material where a lot of different amino acids and fatty acids can be produced
So like purines, porphyrins, haem, chlorophyll, amino acids
Supports the entire width of metabolism that is needed to generate a proliferative phenotype
K-Ras mutations transform the food source
K-Ras V12 induces tumour cells to increase uptake of external protein
This can then be used to directly generate new proteins, ATP and DNA/RNA
Pancreatic cancer- both TP53 and K-Ras mutations
K-Ras mutations provide increased glycolysis through AKT activation
Also increase ability to scavenge extras food from the environment
TP53 mutations increase anabolism and protection against oxidative stress and other toxins
Familial cancer syndromes illustrate role of metabolism in cancer
Mutations in succinate dehydrogenase results in paraganglioma and pheochromocytoma
Mutations in fumarate hydratase result in leiomyoma and renal cell carcinoma
Both of the above enzymes are intermediate reactions in the TCA cycle
Both metabolites disrupt normal Cell signalling processes
Blockages in fumarate hydratase or succinate dehydrogenase will lead to the accumulation of either fumarate or succinate
This leads to HIF activation because both fumarate and succinate are inhibitors of the alpha-ketoglutarate dependent deoxygenases which hydroxylate HIF which leads to its degradation
So the inhibition of this leads to HIF activation and therefore activation of increased glycolysis, cell proliferation and survival
Can the immune system protect the host from cancer
1890s: William Coley treated cancer patients with bacterial extracts (Coleys toxins) to activate general systemic immunity
1950s: using rodent models, it was relatively easy to immunise against transplantable tumours and “tumour immunology” was an “optimistic” field. However this was an artefact of allogeneic responses
Second half of 20th century: experiments by Ludwik Gross, George Klein and more recently Thierry Boon demonstrated that a protective response can be generated against a “non-immunogenic” murine tumour
Immunogenicity of chemically induced tumours
Injecting a chemical carcinogen in a mouse which will induce growth of tumour in the mouse
If you then take the tumour and grow its cells in the lab you can challenge a naive mouse with those cells and it will also develop a tumour
However if you first irradiate some of the tumour cells so they cant proliferate use them to vaccinate a naive mouse, then when the same mouse is challenged with live tumour cells the mouse remains tumour free
This shows tumours can be immunogenic
If you induce tumour growth in a mouse then surgically resect the tumour
You can then culture tumour cells in lab to generate tumour cell line
If these tumour cells are injected into a naive animal they will develop into a tumour but if you inject them into original host from which they had been isolate from the animal remains tumour free
-this demonstrates tumour can induce immunological memory that continues to protect the host overtime
In these types of experiment tumour protection is not seen in T cell deficient mice but it can be conferred by adoptive transfer of T cells from immune mice- suggesting T cells play a key role in
Evidence of tumour protective immunity in humans
Immunosuppressed individuals more frequently develop cancer (especially virus-associated cancers) than immunocompetent individuals
The presence of immune cells within some tumours correlates with improved prognosis eg colorectal cancer
-patients with colorectal cancer usually classified in stages 1-4 based on appearance of tumour and whether lymph nodes involved and whether there’s metastasis
-another study classified based on presence or absence of memory T cells within their tumours
-this proved to be a better indicator of prognosis since it could identify patients with stage 1 disease who wouldn’t survive very long
Cancer immunosurveillance
This concept originally proposed by Burnett and Thomas predicts that the immune system can recognise precursors of cancer and in most cases destroy these precursors before they become clinically apparent
This theory was controversial because early studies in “immunocompromised” mice did not support it
More recent work with fully immunosuppressed mouse models indicates that the theory is correct
However, the immune system not only protects the host from tumour development but also sculpts or edits, the immunogenicity of tumours that may eventually form. Therefore a new term was introduced by Robert Schreiber: cancer immunoediting
The three Es of cancer immunoediting
Elimination: immune mediated destruction of most cancer cells
Equilibrium: dynamic equilibrium between the immune system and any tumour cell variant that has survived the elimination phase. The immune system is enough to contain, but not fully extinguish these genetically unstable and mutating tumour cells “A crucible of Darwinian selection”
Escape: tumour cell variants selected in the equilibrium phase now grow out in an immunologically intact environment (ie the immune system can no longer control it)
Immunoediting of tumours
Tumour cells from immunocompetent mouse have been sculpted by the immune system and are less immunogenic
Tumour cells from immunodeficient mouse were not edited and are more immunogenic and rejected by immunocompetent mice
How might T cells detect and destroy cancer cells
T cells recognise antigens displayed on the cell surface in the form of short peptide fragments bound to molecules encoded by the major histocompatibility complex MHC via T cell receptors
-TCR is a heterodimeric transmembrane structure comprising of alpha and beta chain
T cells can recognise antigen from all cellular compartments (including inside target cell) so they’re broader than antibodies (target antigen only at the cell surface)
Both CD4 and CD8 bind a cytoplasmic tyrosine kinase Lck through the cytoplasmic tail of the a chain and bring it into close proximity with the T cell receptor. The presence of the CD4 or CD8 molecule increases the sensitivity of T cells to antigen presented by MHC molecules by ~100 fold
Activation of Naive T cells requires two independent signals
When the T cell receptor engages its specific MHC complex it delivers signal into the T cell called signal 1 to activate this cell
However a naive T cell is only fully activated if it also receives a second co stimulatory signal
-eg from the interaction of CD28 (expressed on surface of T cell) binding to CD80/CD86 on the target cell
Costimulatory/inhibitory checkpoints regulate T cell recognition
Costimulatory checkpoints stimulate T cell functions including priming and effector responses
Inhibitory checkpoints attenuate T cell functions via checkpoint receptors and ligands expressed by T cells themselves, DCs and other immune cells and tumour cells
Tumour associated antigens
Mutated self proteins (eg from DNA damage):
-cyclin dependent kinase (cell cycle regulator) in melanoma
-B-catenin (signal transduction) in melanoma
-caspase 8 (apoptosis regulator) in squamous cell carcinoma
-KRAS in many tumour types
Aberrantly or overexpressed self proteins:
-oncofoetal antigens (normally expressed in embryogenesis) eg CEA, AFP
-cancer-testis antigens HER2, EGFR
-telomerase (telomere maintenance) in many cancers
Lineage specific (differentiation) antigens:
-Mart1/Melan A, gp100 and tyrosinase in melanoma
-CD19 or surface immunoglobulin in B cell lymphomas
-CD33 or myeloid cells
Abnormal post-translational modification of self protein:
-MUC1 (mucin) overexpressed in an underglycosylated form in breast and pancreatic cancer
Viral proteins (20%of tumours carry a virus):
-HPV in cervical cancer
-EBV in Hodgkin’s lymphoma, nasopharyngeal carcinoma
What makes a good target antigen for tumour immunotherapy
Tumour-specific
-reduced toxicity
Shared amongst patients with the same and different tumour types
-widely applicable
Critical for tumour growth/survival
-lack of antigen-loss variants
Lack of immunological tolerance
-high avidity T cells
Central tolerance occurs in the thymus
Occurs during T cell development in the thymus
In the thymus, as the T cells each express T cell receptor they go through 2 rounds of selection
-within the cortical epithelium they are subject to positive selection to see whether T cell receptor can interact with MHC molecule. If it cant then those T cells are actively destroyed though programme cell death
-the TCR is then subject to a second round of selection called negative selection to test to see if it reacts to a self antigen
-this could be dangerous as it could induce autoimmunity so it is deleted by apoptosis
So the only T cells that should be released from thymus as those capable of seeing antigen but dont see your own tissue antigen
Peripheral tolerance
Any potential auto-reactive T cell that escapes the thymus will be inactivate when it engage its target antigen on your own tissue in the absence of co-stimulation
Such a T cell undergoes anergy so that it will no longer respond to that antigen even when presented by an APC like dendritic cell
What makes a good target antigen for tumour immunotherapy
Tumour specific (reduced toxicity):
-good examples: mutated self proteins, viral antigens, cancer-testis antigens
-poor examples: lineage specific antigens, overexpressed self proteins
Shared amongst tumours:
-good examples: mutated self or viral proteins involved in oncogenesis, lineage specific antigens
-poor examples: mutated self proteins incidental to oncogenesis
Critical for tumour growth/survival (lack of antigen-loss variants):
-good examples: mutated self or viral proteins involved in oncogenesis. Overexpressed self proteins (telomerase)
-poor examples: mutated self proteins incidental to oncogenesis, lineage- specific antigens
Lack of immunological tolerance (high avidity T cells):
-good examples: mutated self proteins, viral antigens
-poor examples: lineage-specific antigens, overexpressed self proteins
MHC class I processing pathway
All proteins synthesised in cytosol of cell
A proportion of each of these cells will be broken down by the proteosome into small peptide fragments
These fragments are actively pumped into the ER by the TAP transporter where they can associate with newly formed MHC molecules
If the peptide fragment contains appropriate sequence of AA to fit in peptide binding groove of MHC then it will form a stable structure that buds out of the ER and travels to cell surface where it can be engaged
-partly folded MHC class I alpha chains bind to calnexin until B2-microglobulin binds
-MHC class I a:b2m complex is released from calnexin, binds a complex of chaperone proteins (calreticulin, Erp57) and binds to TAP via tapasin
-cytosolic proteins and defective ribosomal products DRiPs are degraded to peptide fragments by the proteasome. TAP delivers peptides to the ER
-a peptide binds the MHC class I molecule and completes its folding. The MHC class I molecule is released from the TAP complex and exported to the cell membrane
Cell surface molecules of the immunoglobulin superfamily are important in the interaction of lymphocytes with APCs
When a TCR engages with APC/tumour cell this interaction is stabilised with molecular interactions involving adhesion molecules such as LFA-1 on surface of T cell engaging I-CAM 1 and 2 on target cell
If there is interference with any of this mechanism or in the MHC processing pathway then T cells may no longer be able to detect the tumour cell
Mechanisms whereby tumours might escape the immune response
-loss of HLA class I expression (eg lung cancer, prostate, melanoma etc)
-reduced expression of other molecules involved in antigen processing/presentation (TAP1, LMP2, LMP7 and tapasin) (eg colorectal cancer)
-loss of costimulatory molecule expression (eg CD80, CD86)
-loss of adhesion molecule expression (eg ICAM1)
-loss of target antigen (eg melanoma)
-inhibiting T cell infiltration
—endothelin B receptor expression on the tumour endothelium signals to prevent modulation of ICAM and thereby reduces T cell adhesion to the vascular endothelium
—nitrosylation of chemokines can keep T cells from entering the tumour core
—fibrosis sequesters T cells away from tumour
Mechanisms whereby tumours might escape the immune response continued
Immunosuppression at the tumour site:
-transforming growth factor B- TGFB suppresses anti-tumour T cell functions and induces Tregs
-Indoleamine 2,3-deoxygenase-IDO expressed by tumours catabolises tryptophan and can block CD8+ T cell proliferation and promote apoptosis of CD4+ T cells (eg prostate, colon cancer)
-factors that inhibit differentiation, maturation and function of local DCs (eg VEGF, IL-6, IL-10, TGFa, M-CSF, NOS2, arginase-1, IDO, PGE2, COX2 and gangliosides) such that DCs mediate immunosuppressive effects and promote Treg differentiation
-CD95L expression by the tumour endothelium or by locally activated T cells might induce the death of CD95-expressing tumour specific T cells
Immunosuppression at the tumour site: Tregs
Regulatory T cells- functionally defined by their ability to inhibit an immune response by influencing the activity of another cell type:
-CD4+
-FoxP3+ (transcriptional repressor)
-CD25+ (IL2 receptor on a chain)
-CD127- (IL7 receptor alpha chain)
Mediate function through cytokine release (eg IL10, TGFB) and/or direct cell-cell contact
Important in controlling immune responses to self antigens
May play a role in tumour escape (Hodgkins lymphoma, ovarian cancer)
May be selectively recruited to the tumour site by chemokines (eg CCL22 recruits CCR4+Tregs to ovarian cancer)
Immunosuppression at the tumour site: myeloid-derived suppressor cells (MDSC)
A heterogenous population of cells of myeloid origin that comprises myeloid progenitor cells and immature macrophages, granulocytes and dendritic cells
Expand during cancer, inflammation and infection (patients with different types of cancer can carry 10x as many MDSCs in their blood as health individuals)
Potent suppressors of T cell function
Show upregulated expression of immune suppressive factors such as Arginase-1 and inducible nitric oxide synthase (iNOS) both of which metabolise L-Arginine. The shortage of L-Arginine inhibits T cell proliferation. INOS also generates nitric oxide which inhibits T cell function and induces apoptosis
May induce Tregs
Inducing T cells for cancer therapy
Non-specific T cell stimulation
Vaccination
Adoptive T cell therapy
Non-specific T cell stimulation
Immunostimulatory cytokines eg IL-2 for melanoma and renal cell carcinoma
-IL2 is a T cell growth factor so by injecting it you can boost T cell response against the tumour
Blockade of immunologic checkpoints. Eg administering antagonistic antibodies Ipilimumab specific for the coinhibitory receptor CTLA-4 improved overall survival in patients with previously treated metastatic melanoma
-CTLA-4 outcompetes CD28 for binding of CD80/CD86 stops T cell response from running out of control
Vaccination
Vaccines currently being tested:
-tumour cells/lines (irradiated or lysates)- may be combined with strategies designed to enhance the immunogenicity of the tumour cells (eg induce expression of costimulatory molecules CD80/CD86 and 41-BBL or immunostimulatory cytokine GM-CSF)
-dendritic cell based vaccines eg sipuleucel-T (Provenge) used to treat metastatic prostate cancer (DCs and prostatic acid phosphatase and GMCSF)
-defined peptides or protein antigens (purified or expressed from recombinant viruses, DNA or RNA)
Adoptive T cell therapy
Infusing whole T cell populations (eg donor lymphocyte infusion for CML patients who relapse after allogeneic BMT)
Infusing selected, tumour specific T cells
Activate T cells more effectively in vitro
Fully characterised T cells
Achieve very high frequencies of reactive T cells in vivo
Supports development of vaccination strategies
Melanoma - TIL therapy
It’s easy to biopsy melanomas- skin cancer
Tumour biopsies contains relatively large number of tumour infiltrating lymphocytes TIL
Can then select tumour specific TILs by stimulating them with melanoma cells lines
These TILs can be expanded in lab-> generated 10^10 from a single patient
These cells are then infused back into patients bloodstream and to aid the engraftment and expansion of T cells, IL-2 is given
In advance of T cell infusion, patients often receive non-myeloablative conditioning using drugs such as cyclophosphamide and fludarabine
-these drugs deplete the hosts haemopoeitic system creating space and increasing cytokines that encourage expansion of infused T cells
-it may also help to deplete numbers of immunosuppressive regulatory T cells in the patient
Engineering T cells for cancer therapy: TCRs or chimeric antigen receptors CARs
If you can isolate a single cancer specific T cell from one patient then you can clone the genes that encode for TCR
These TCR chains determine what antigen the T cell responds to
So after cloning the TCR genes they can be packaged into a retrovirus or lentivirus vector which can be used to deliver genes in the lab into T cells of a cancer patient where they will integrate into host cell chromosome
When the genes enter the cell they encode for the same TCR so now these cell can be returned to patient to target cancer
Similar method for CARs
CAR-T cells are engineered to express receptors specific for tumour antigens
Chimeric antigen receptor
Combines:
-the target specificity of an antibody
-the potent cytotoxic and immune regulatory activity of a self replicating T cell
-MHC unrestricted
-targets any surface molecule (even non proteins)
Challenges:
-requires autologous T cells, identification of tumours antigens
-so far CD19 most successful CAR target
Engineering “smarter” immune cells: CAR-T therapy
Relapsed, refractory all patients treated
Complete remission in 90% patients
Very effective tumour clearance
Long-lasting responses… potential cures
“On target” side effects can be severe
Autoimmunity
Studies in mouse models suggests that potent tumour immunity can be induced by vaccination with specific self antigens or unfractioned tumour derived material with little or no evidence of autoimmunity
TIL therapy resulted in symptoms of autoimmune melanocyte destruction including vitiligo or uveitis in some melanoma patients
Administering antagonistic antibodies specific for CTLA-4 or PD-1/PD-L1 was associated with severe inflammatory responses in up to 1/3 of patients (checkpoint blockade toxicity) ranging from dermatitis to severe chronic colitis or acute hepatitis
Monoclonal antibodies for cancer therapy
Immune mediated mechanisms:
-blocking actions of immune checkpoint molecules
-complement mediated lysis (eg rituximab (mabthera)- antibody binds to its target antigen and then activates the complement cascade which involves a series of proteolytic event resulting in the formation of a membrane attack complex which punches hole in target cell
-antibody dependent cellular cytotoxicity ADCC: antibody binds then recruits NK cell which engages Ab through binding via fc-y-RIIIa on its surface
Direct effects:
-blocking receptor: ipilimumab binding CTLA4 and blocking immune checkpoint. Erbitux and herceptin inhibit binding of ligands to HER1 or HER2 which blocks proliferation of HER1 or HER2 expressing cells through inhibition MAPK/PI3K/AKT pathway. Both also induce apoptosis
-anti-angiogenesis: Avastin is directed against VEGF and blocks its interaction with its receptors and was therefore inhibit angiogenesis. Erbitux and herceptin inhibit VEGF production
-cytotoxic effect of conjugate antibodies: antibody bound to radionuclide- radiation reaching target cell induces DNA damage and apoptosis
—antibody bound to protein toxin molecule
—antibody bound to a cytotoxic drug
