principles of cancer biology 22% AB Flashcards
What is the normal function of proto-oncogenes?
normal genes in the body that help cells grow and divide to make new cells
List the regulatory levels that can be altered to result in proto-oncogene oncogenesis to an oncogene?
1) genomic mutations: point mutations, insertions/deletions, inversions, duplications, translocations
2) epigenetic changes: specific, global
3) proteomic changes: ubiquitination, viral oncoproteins
4) metabolic changes
What is an oncogene?
a gene which in certain circumstances can transform into a tumor cell
List key oncogenes
p110a
EGFR
ERBB2 (HER2)
B-RAF
K, H, N - RAS
MYC
BCR-ABL = RALEIGH CHROMOSOME in dog
IDH 1/2
JAK2
KIT
MET
FLT-3
ALK
ROS1
NTRK
Example of a proto-oncogene?
MDM2
*AG study guide
Trastuzumab is a monoclonal Ab that targets which receptor/oncogene?
HER2 (ERBB2) receptor, HER2/Neu oncogene
Which chromosome is involved in BCR in humans? Dogs? ABL?
Humans = chromosome 22
Dogs = chromosome 26
ABL = chromosome 9 in BOTH species
What is the result of BCR-ABL fusion at the cellular level?
constitutively activated tyrosine kinase in the cytoplasm –> continuous cell growth
Which drug inhibits BCR-ABL?
Imatinib (Gleevec) - binds near ATP binding site –> locks BCR-ABL in an inhibit conformation
What type of translocation occurs in human CML?
BCR-ABLR (chromosome 9 and 22 translocation)
*reported in ALL and AML
Which molecular alterations are associated with canine LSA?
- Myc-IgH translocation in BL
- Rb deletion in CLL
- Bcr-Abl translocation in CML
List the MYC targets and promotion of tumorigenesis
- promotes cell cycle progression
- down regulation of CDK inhibitors
- up regulation of cyclin D1, CDk4 CDC25A and E2F transcription factors
What is the most common mechanism of MYC deregulation?
whole genome doubling or tandem duplications
Which mutations can inhibit then promote cancer?
myc, TGFb
Which chromosome is c-myc located on in people?
chromosome 8
*regulates 15% of all genes
List the genes related to sustained proliferative signaling.
Ras, Myc
Which genes are involved in evading growth suppressors?
p53, Rb1, PTEN, CDKN2A (p16)
Which genes induce angiogenesis?
VEGF-a, VHL
Which genes are involved in activating invasion and metastasis?
N-cadherin, SNAIL, Slug, TWIST, Zeb 1/2
Which genes resist cell death?
Bcl-2 and Bcl-XL upregulation
Suppress Bax, Bim, Bak
Which genes enable replicative immortality?
TERT, wnt, Notch, Hh pathway
Which genes deregulate cellular energetics?
Warburg effect, GLUT1 transporters, Rad, Myc, p53 mutants
Which genes are affected with genomic instability and mutations?
mutations in “house keeping” genes
Which genes are involved in tumor-promotion/inflammation?
GFs, proangiogenic factors
Which kinases are monomeric?
EGFR, FGFR
Which kinases are dimeric?
PDGFR
Which receptors does epidermal growth factor (EGF) bind?
ERBB1, 3, 4
ERBB2 = HER2 but does not bind directly (dimerizes with other ERBBs)
Which portion of the EGF/ERBB is subject to oncogenic mutations?
all 3 portions (N terminal extracellular ligand binding domain, intracellular kinase domain, and C regulatory tail)
*transmembrane receptors that signal intracellular downstream
In which tumor type is ERBB2 over expressed?
mammary carcinoma
Draw the PI3 kinase Pathway
Where do alterations occur in the PI3K pathway in cancer?
- PTEN loss
- p110 activating mutations/amplifications
- less frequent p85, AKT, PDK genes
In the PI3K pathway, what does PIP3 phosphorylate?
AKT
What mutation occurs in ~50% of canine HSA?
PTEN –> mTOR/PI3k pathway disruption
DLBCL in dogs and humans share which common pathway mutations?
NFKB and PI3K, Jak Stat (n-ras, p53, Rb, p16, CDK, telomerase)
Ras cell signaling is associated with multiple types of human cancers. Which hallmark does Ras signaling best represent?
Sustained proliferative signaling
Which pathway is RAS and RAF involved? Draw it.
MAP kinase pathway
RAS GDP bound = inactive, GTP bound = active
List the 3 RAS genes and the most common mutations
K-RAS, N-RAS, H-RAS
gain of function substitutions in codons 12, 13, and 61 which impair RAS-GTP hydrolysis locking the protein in an activated state
What is the primary downstream effector of RAS?
RAF
B-RAF = V600E substitution in people = V595E in DOGS
Which percentage of canine UC have RAF mutations?
87% V595E somatic mutation on chromosome 16
How might you treat a patient that develops resistance to a RAF inhibitor?
MEK inhibition
- Upstream: Farneyl transferase inhibitor, gerangyglgeranyl transferase inhibitor (AG SG)
Name of BRAF inhibitor?
vemerafinib
Name of MEK inhibitor?
Trematinib “MEKinist”
List the proangiogenic oncogenes.
RAS, HER2, EGFR
List the antiangiogenic oncogenes.
PTEN, p53, VLH
Role of IDH1/2?
homodimeric enzymes that catalyze the conversion of isocitrate to alpha kteoglutarate resulting in NADPH from NADP+
Mutations found in HUMAN AML, chondrosarcoma, glioma, and cholangiocarcinoma
Define tumor suppressor genes.
normal genes that slow down cell division or result in apoptosis. When ineffective, cells grow out of control.
List the key tumor suppressor genes.
p53
PTEN
p16 (ink4a)
p14 (arf)
BRCA 1/2
LKB1
VHL
APC
FBXW7
NF 1/2
Which cancer has mutations in STAT3 and p53?
OSA
p53 defects are associated with which disease?
Li Fraumeni - rare autosomal dominant disorder that leads to a wide variety of cancers
What regulates p53 and how does affinity change based on phosphorylation?
- MDM2
- when p53 is phosphorylated by ATM its affinity for MDM2 is reduced which allows p53 to accumulate and act as a transcription factor
Which is the most frequently mutated gene in human cancer?
TP53 gene encoding p53 protein
Where is p53 normally located? Following stress signals?
Cytoplasm then moves to nucleus
In which phase of the cell cycle does p53 cause arrest?
G1
What activates intrinsic apoptotic pathway?
cell stress, p53
What is the most common mechanism of loss of heterogeneity?
mutations in p53 or other tumor suppressor genes
PTEN associated chromosome?
10q23 (humans)
*phosphatase and tensin homologue deleted on chromosome TEN
What is the function of PTEN normally?
- dual specificity protein and lipid phosphatase- blocks PI3k signaling by inhibiting PIP3 dependent AKT activation therefor inhibiting cell survival, growth, and proliferation
- Converts PIP3 back to PIP2
- inhibits oncogenic transformation
What happens with PTEN down regulation/loss?
increased PIP3 levels –> hyper activation of downstream AKT signaling –> cell growth/proliferation/etc
What is the most common mechanism of PTEN loss?
chromosomal deletions at 10q23 locus
Mutations in BRCA 1/2 result in?
breast and/or ovarian cancer in people (heredity)
Mutations in BRCA 2 or Fanc have been associated with which disease?
Fanconi anemia
List the caretaker genes, their function, and tumor risk if mutated.
- BRCA 1, DNA repair, breast/ovarian cancer
- BRCA 2, DNA repair, breast cancer (female or male)
- MSH2/1, DNA mismatch repair, hereditary non polyposis colorectal cancer
List the gatekeeper genes, their function, and tumor risk if mutated.
- p53, transcription factor, Li-fraumeni syndrome
- RB1, transcription regulator, familial retinoblastoma
- APC, regulates B-catenin function, familial adenomatous polyposis
BRCA 2 and RAD 51 are needed for what repair mechanism?
homologous recombination
What is RAD 51?
- ATPase that forms a nucleoprotein filament on single-stranded DNA.
- function of finding and invading homologous DNA sequences to enable accurate and timely DNA repair
In which scenario would PARP inhibition be effective?
if compromised dsDNA repair (e.g. with BRCA 1/2 loss or RAD51 loss)
When tumors lack or have defective BRCA 1/2, what is the result?
- not able to use homologous recombination for DNA repair
- use instead PARP as an alternative repair pathway (why PARP inhibition is effective, blocks ssDNA repair)
What occurs when giving a PARP inhibitor with platinum agent or RT to patients with BRCA mutations?
- synthetic lethality
- BCRA mutations = homologous recombination defect, dependent on base excision repair
- PARP inhibitors impair base excision repair –> cell death
Name of PARP inhibitor?
Olaparib
Describe homologous recombination. Acts for which type of DNA? In which phase of the cell cycle?
- type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA
- dsDNA
- G2
What is the normal function of the retinoblastoma protein (Rb)?
- tumor suppressor that regulates activity of transcription facts in the E2F family constraining cell cycle progression at the G1 to S phase transition
- when phosphorylated does not bind E2F –> cell cycle progression
- when hypophosorylated binds E2F –> stops cell cycle progression
Which viral cancer is Rb associated with?
HPV in humans
- E7 protein bind to Rb causing phosporylation and progression through cell cycle for duplication
Define single nucleotide polymorphisms (SNP).
genomic variant at a single base position in DNA
e.g. “ATCGA” sequence replaced “ATTGA”
- introns or exons
- happen frequently
Define copy number variant (CNVs).
occurs when the number of copies of a particular gene varies from one individual to the next
single nucleotide polymorphisms role in cancer?
- can occur as driver mutations (e.g. affect critical gene)
- influence gene expression
- may occur in promoter regions
- may effect genes involved in mismatch repair and cell cycle regulation
List the types of (copy number variation) CNVs
- Deletion: segment of DNA missing
- Duplication: one or more extra copy of DNA
- Insertion: additional segment of DNA inserted into the genome
- Inversion: segment of DNA flipped or reversed in the genome
- Translocation: segment of DNA moved from one area to the next
What is the affected pathway for ataxia telangiectasia and target protein?
DNA double strand break response; ATM
Nijmegen breakage syndrome has what defective pathway?
DNA double strand break response
How does p53 cause cell cycle arrest?
ATM stabilize/phosp p53 when DNA DSB, p53 activates p21 stops G1/S checkpoint
How does DNA variation (e.g. copy number variants and DNA breaks) result in cancer?
variations cause changes in AA sequences of encoded proteins which may result in hyper activation of oncoprotein or inactivation of tumor suppressors
What are the mechanisms of loss of heterogeneity?
gene conversion, translocation, mitotic recombination
What are 4 ways MYC is activated?
Retrovirus transduction, amplification, translocation, proviral insertion
Define internal tandem duplication.
genetic mutation characterized by the insertion of a segment of DNA resulting in duplication of the gene in tandem/adjacent to the original segment
Which is the most common gene affected by tandem duplication?
MYC
Which missense mutations have been found in dog AML?
FLT3, Ckit, RAS
What is FLT-3?
FMS-like tyrosinase kine 3 receptor = member of the PDGFR family
*AML +/- polycythemia vera (cannot confirm)
In which cancer is FLT3 expressed in both humans and dogs?
AML
- 25-30% of AML in people have FLT3 mutations (internal tandem duplications or point mutations) –> on-going activation of MAPK
Which receptors does toceranib target?
KIT, PDGFR, VEGFR-2, flt-3
Somatic vs germline mutations
Germline mutations are inherited occurring in germ cells where somatic mutations occur in the individual secondary to DNA replication error or environmental exposure in somatic cells
What is one of the most common somatic mutations in canine cancer?
V595E in dogs with UC on chromosome 16 (exon 15)
Li Fraumeni syndrome occurs in people with germline mutations. What type of germline mutations?
autosomal dominant, P53
Genetic inheritance of single nucleotide polymorphisms.
- somatic copy number variants are known to be mutated in multiple human cancers (lung, glioblastoma, breast, colorectal)
- less well documented but heritable germline mutations in copy number variants may also contribute to cancer (neuroblastoma, glioma)
Which breeds have germline mutations in BRCA1 predisposing to mammary cancer?
Shih tzus, English springer spaniels
Which germline/testis protein is expressed in oral melanoma?
MAGE-A (81%)
*also nasal tumors and TVT (100% both),
What is MEN1 and 2?
- Multiple neuroendocrine neoplasia 1 and 2 resulting from germline mutations in tumor suppressor
- Men2 autosomal dominant in RET (rearranged during transfection)
List the key differences between oncogenes and tumor suppressor genes.
oncogenes:
- altered versions of proton-oncogenes that play a role in promoting cellular proliferation “gas pedal” to cancer
- mutations at the cellular level causes a DOMINANT GAIN OF FUNCTION
- occur in SOMATIC CELLS
tumor suppressor genes:
- genes involved in blocking cellular proliferation “brakes” in cancer
- mutations are RECESSIVE and require loss of both alleles for phenotypic change
- LOSS OF FUNCTION
- can occur in GERM CELLS (inherited cancer predisposition) and/or can arise in SOMATIC CELLS
What does epigenetic extrachromosomal elements refer to?
genetic material outside of chromosome in the cell that can have an impact on DNA/RNA expression.
e.g. complimentary DNA, long non coding RNA, RNA interference, silencing RNAs
What is complementary DNA (cDNA)? Use?
synthetic DNA transcribed from mRNA through use of the enzyme reverse transcriptase
mostly in lab for cloning, molecular probes, gene expression studies
What are long non-coding RNAs? Impact?
- RNA molecule longer than 200 nucleotides that DOES NOT code for proteins
- can influence gene regulation interacting with DNA, RNA, or proteins acting as scaffolds or sequestering proteins away from target genes.
- have epigenetic influence on chromatin structure and modification
- can influence transcription and cell cycle
- Implicated in disease
Which veterinary cancers have been associated with dysregulated long non-coding RNAs?
- OSA: MALAT1 and HOTAIR RNA
- oral SCC feline
- equine sarcoids
- k9 LSA
- STS
- mammary tumor
(summary article Vet Sci 2023 Bennett [probs not that important])
RNA interference - what is it?
- process of mRNA degradation that regulates the activity of genes by silencing or suppressing their expression via small interfering dsRNA (siRNA)
How does RNA interference work?
- Production of Small RNAs: include microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs are transcribed from genes in the cell’s genome and processed into mature miRNAs, while siRNAs can be derived from exogenous sources such as viruses or introduced experimentally.
- Loading onto RNA-Induced Silencing Complex (RISC): The small RNAs are then loaded onto a protein complex called the RNA-induced silencing complex (RISC). Within the RISC, the small RNA serves as a guide sequence that recognizes complementary sequences in target mRNAs.
- Target mRNA Binding: The RISC complex with the small RNA guide sequence binds to target mRNAs through base-pairing interactions. This binding occurs primarily in the 3’ untranslated region (UTR) of the target mRNA.
- Silencing of Gene Expression: Once bound to the target mRNA, RNA interference can lead to gene silencing
What are the two main mechanisms of RNA interference gene silencing?
- mRNA degradation - binding to RNA-induced silencing complex(RISC) to target mRNA leads to degradation of mRNA molecule by cellular enzymes preventing it from being translated into a protein
- translation inhibition - RISC complex interferes with the ribosomes ability to initiate protein synthesis rather than direct degradation
What produces small interfering RNAs (siRNA)?
DICER - key enzyme responsible for producing micro and small interfering RNAs from precursor RNA molecules
Describe how DICER works on the RNA interference pathway.
- Precursor RNA Processing: DICER acts on precursor RNA molecules, which can be long double-stranded RNA (dsRNA) molecules or hairpin-loop structures formed by single-stranded RNA.
- Cleavage by DICER: DICER cleaves the precursor RNA molecules into small RNA duplexes typically around 20-25 nucleotides in length. These small RNA duplexes consist of two strands: a guide strand and a passenger strand. The guide strand, which is usually the one with the less stable 5’ end, is loaded onto the RNA-induced silencing complex (RISC) for target mRNA recognition and gene silencing, while the passenger strand is typically degraded.
- Loading of Guide Strand onto RISC: DICER interacts with other proteins to facilitate the loading of the guide strand of the small RNA duplex onto the RISC complex. The guide RNA serves as a sequence-specific recognition element that guides the RISC complex to complementary sequences in target mRNAs.
- Target mRNA Silencing: The RISC complex, loaded with the guide RNA, binds to target mRNAs through base-pairing interactions, leading to gene silencing through mRNA degradation or translational inhibition.
What is the consequence of micro (miRNA) formation?
Activates RISC enzyme
What are the differences between RNA silencing with micro RNA (miRNA) and small interfering RNA (siRNA)?
- siRNA is EXOGENOUS and specifically binds to mRNA target
- miRNA is ENDOGENOUS with less binding affinity
How are RNA included silencing complex (RISC) and micro (miRNA) related?
- mature miRNA part of active RISC containing DICER
What is the pathway of micro (miRNA) synthesis?
introns and/or other non coding areas (pri-miRNA) produced in nucleus –> precursor miRNA by Dorosha –> miRNA by DICER –> miRNA activates RISC by inhibiting complementary RNA –> neg feedback on DNA transcription
What may be the result of deregulation in DICER and RISC of the RNA interference pathway?
uncontrolled protein synthesis/lack of degradation
What is RNA silencing?
sequence specific cellular responses to RNA (including RNA interference and others) that plays a critical role in regulation of cell growth and differentiation using ENDOGENOUS small RNAs (miRNAs) that play role in carcinogenesis
What exports pre-RNA from the nucleus to the cytoplasm?
Exportin 5
What are telomeres?
structures at the end of chromosomes that consist of repeating nucleotide sequences
What is the function of telomeres?
- function to protect chromosomes from deteriorating (losing important DNA info during replication) or fusing with other chromosomes
- maintain genetic stability
Describe the structure of a telomere
2 main parts:
1) telomere sequence that forms cap at end of telomeres 5’TTAGGG3’
- Forms T-loop when unwound for replication
2) associated proteins that stabilize structure and regulate length
- Shelterin
- telomerase
- DNA repair proteins
- Check point proteins
What happens to telomeres as cells divide?
gradually shorten - once critically short cells become senescent or undergo apoptosis
*telomere length implicated in many degenerative/aging diseases
How much faster do dogs loose telomeric DNA compared to human?
10x faster
How do cancer cells protect their telomeres?
Telomerase activation - adds new telomere DNA onto chromosome ends or ALT
What is the role of the sheltering complex? Associated proteins?
- bind directly to telomeric DNA to protect from degradation
- TRF1 (Telomere Repeat-Binding Factor 1)
- TRF2 (Telomere Repeat-Binding Factor 2)
- POT1 (Protection of Telomeres 1)
- TIN2 (TRF1-Interacting Nuclear Factor 2)
- TPP1 (Adenine Thymine-rich - Telomere Protein Component 1)
- RAP1 (Repressor Activator Protein 1)
Role of telomerase and components?
- enzyme that adds telomeric DNA sequence
- TERT (Telomerase Reverse Transcriptase): The catalytic subunit responsible for synthesizing telomeric DNA.
- TER (Telomerase RNA Component): Provides the template for the addition of telomeric repeats by TERT.
What is telomerase effect on cell signaling pathways?
WNT and B-catenin
- WNT/B-catenin regulates telomerase by increasing TERT
- without B-catenin you get shorter telomeres
- cancer can upregulate B-catenin
Where are the 3 major cell cycle check points?
end of G1
End of G2
Metaphase
What indirectly regulates CDKs?
p53
What needs to be present to proceed through the cell cycle?
growth factors and mitogens
Which cyclin drives G1/S phase transition?
Cyclin D
What happens when cyclin D binds CDK 4/6?
partially phosphorylates Rb –> E2F activates transcription of cyclin E gene –> fully phosphorylates Rn –> E2F inactivation
What does cyclin E phosphorylate?
Rb and p27/KIP1 –> cyclin D inhibitor
Degradation of p27/KIP1 promotes?
expression of cyclin A
What promotes transcription of cyclin A? How?
E2F by removing repressor molecule cell cycle responsive element (CCRE) from promoter allowing cell to enter S phase
Which cyclin plays a role in 2 stages?
cyclin A
cyclin A/CDK2 –> progress to S phase –> resides in nucleus –> initiation/completion of DNA replication
cyclin A/CDK1 –> entry to M phase
Which cyclin ensures there is only 1 DNA copy per cell cycle?
- cyclin A by preventing assembly of excessive replication complexes
- replaces cyclin E when binding to CDK2 –> cyclin A/CDK2 reaches threshold –> terminates assembly of pre-replication complex
Which cyclin forms pre replication complex?
cyclin e
*cyclin a terminates
Role of cyclin A/CDK 1 complex?
stabilizes cyclin B/CDK1 complex then becomes ubiquitinated –> induces mitotic exit/end of mitosis
Cyclin B + CDK1 forms?
maturation/mitosis promoting factor
High concentrations of which cyclin are required to enter M phase?
Cyclin B
*low concentrations to exit M phase
Graph cyclins concentration and cell cycle
Cyclin-Dependent Kinase Inhibitors (CKIs)
- CKIs are proteins that inhibit the activity of cyclin-CDK complexes, acting as negative regulators of the cell cycle
- INK2
- cip/kip
INK2 family inhibit? Examples?
- inhibit CDK4 and 6
- p16, p15, p18, p19
Cip/kip examples?
- cyclin dependent kinase inhibitors
- p21, p27, p57
Rb role in cell cycle?
regulates G1 to S phase transition
Anaphase-Promoting Complex/Cyclosome (APC/C)
multi-subunit ubiquitin ligase complex that regulates the metaphase-to-anaphase transition and exit from mitosis
What can be used with flow cytometry to determine cell stage of apoptosis?
Annexin V and propidium iodide (PI)
- through differences in plasma membrane integrity and permeability
What is one of the first steps in apoptosis?
flipping the cell membrane so the phosphatidylserine is exposed outside
What binds phosphatidylserine in apoptosis?
Annexin V with high specificity = marker of early apoptosis
Which is higher in late apoptosis annexin or propidium iodide (PI)?
- PI penetrates through plasma membrane and binds nucleic acids
State stage of apoptosis based on annexin V (AV) and propidium iodide (PI):
AV-/VI-
Viable cell
State stage of apoptosis based on annexin V (AV) and propidium iodide (PI):
AV+/VI-
early apoptosis
State stage of apoptosis based on annexin V (AV) and propidium iodide (PI):
AV+/VI+
late apoptosis
What can be used on flow cytometry to differentiate G0 phase from other phases (G1, S, G1/M)?
ki67 - not expressed in G0
What can be used on flow cytometry to differentiate G0 from G1?
Hoechst 33342 and Pyronin Y
Define growth fraction.
proportion of actively dividing cells within a population at any given time. It represents the fraction of cells that are actively progressing through the cell cycle (G1-M) and undergoing cell division compared to those that are in a quiescent (non-dividing) state (G0).
How can growth fraction be measured?
- Bromodeoxyuridine (BrdU) Incorporation Assay: a thymidine analogue that is incorporated into newly synthesized DNA during the S phase of the cell cycle. After a period of BrdU exposure, cells are fixed and processed for immunostaining using anti-BrdU antibodies. The fraction of BrdU-positive cells relative to the total cell population provides an estimate of the growth fraction.
- Flow: propidium iodine, Hoescht, Ki67
- IHC: proliferation markers
- 3H-Thymidine Incorporation Assay: measures DNA synthesis by assessing the incorporation of radiolabeled thymidine (3H-thymidine) into newly synthesized DNA during the S phase of the cell cycle. The proportion of labeled cells reflects the growth fraction.
High growth fraction biologic behavior?
- rapid growth, aggressive behavior
- also usually more responsive to chemotherapy d/t more rapidly dividing cells
A higher mitotic count will be found in tumors with cells in which phase of the cell cycle?
S and G2 phase entering mitosis
Growth fraction and mitotic count?
High growth fraction is associated with higher mitotic count
What are extracellular growth factors? Structure?
- secreted polypeptide molecules recognized by membrane bound receptors that signal intracellular biochemical signaling responses to control cell properties and proliferation
- polypeptides (monomeric or dimeric), transmembrane protein domain, intracellular kinase domain
Epidermal growth factor (EGF) family - receptors and expression in?
- Epidermal Growth Factor (EGF), Transforming Growth Factor alpha (TGF-α), Epiregulin, Amphiregulin.
- Receptors: Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1).
- Overexpression or mutation of EGFR in lung, colorectal, and head and neck cancers in people. Mammary caricnoma in dogs.
What are examples of split kinases?
VEGFR, PDGFR, FGFR
Which kinases are monomers? Which kinases are dimers?
- Mono: EGFR, FGFR
- Di: PDGFR
Insulin-like Growth Factor (IGF) Family - receptors, expression significance?
- IGF 1, 2
- Receptors: IGFR
- Activation of IGF signaling promotes cell growth and survival in human breast, prostate, and colorectal cancer. IGF-1 feline acromegaly.
Vascular Endothelial Growth Factor (VEGF) Family - receptors, significance?
- VEGF A, B, C, D
- Receptors: VEGFR-1 (Flt-1), VEGFR-2 (KDR,Flk-1), VEGFR-3 (Flt-4)
- VEGF signaling promotes angiogenesis
Human clear cell renal carcinoma is associated with extremely high levels of VEGF, florid angiogenesis, and hypervascularity. This is attributed to high frequency of loss (70%) of function of which gene?
- Von Hippel-Lindau TSG
- Ubiquitin ligase targeting Hif-1a in O2 rich environement
- recessive mutation, LOH common
- Renal clear cell CA due to active HIF
Pro angiogenic factors?
VEGF-a, PIGF, FGF-3, FGF-4, IL-8, IL-6, TNFa
Anti angiogenic factors?
TSP-1, endostatic, angiostatin, tumorstatin, PEX
Which drugs target VEGFR-2?
Toceranib, sorafenib
bevacizumab targets VEGF
What is the role of Tie-1?
- VEGFR and Tie signaling coordinate process of angiogenesis
- RTK expressed on blood vessels
- Involved in recruitment of pericytes and smooth muscle
- maintain vascular integrity
TKIs and targets- palladia?
KIT, PDGFR, VEGFR-2, Flt-3
TKIs and targets - masitinib?
KIT (most specific inhibitor), PDGFR, FGFR3, Lyn
TKIs and targets- imatinib?
ABL (ATP pocket), KIT, PDGFR-a
Platelet-Derived Growth Factor (PDGF) Family - receptors, expression?
- PDGF A, B
- Receptor: PDGFR
- aberrant signaling in gliomas, sarcomas, GIST.
Which receptors are inhibited with martinis?
- lyn = intracellular kinase
- KIT, PDGFR, FGFR
Mechanism of resistance to imatinib?
mutation at ATP-binding site on Abl protein
Fibroblast Growth Factor (FGF) Family - receptor, expression?
- FGF1-23
- Receptors: FGFRs
- Dysregulation of FGF signaling promotes angiogenesis, proliferation, bladder, prostate, and breast cancer.
Hepatocyte Growth Factor (HGF) Family - receptor, expression?
- HGF
- Receptors: Met Proto-Oncogene (c-Met), RON
- HGF/c-Met signaling is involved in cancer cell proliferation, invasion, and metastasis, gastric, liver, and pancreatic cancer.
Nerve Growth Factor (NGF) Family - receptor, expression?
- NGF
- Receptor: Tyrosine kinase receptor A (TrkA), p75 neurotrophin receptor (p75NTR).
- promoting cell survival and metastasis in cancers like neuroblastoma and prostate cancer
- Librela anti NGF moAB
General growth factor signaling cascade
- in the absence of binding in inhibitory state
- Binding of GF or ligand to receptor
- Conformation change to extracellular domain (some already in dimeric forms e.g. PDGFR- others monomers (EGFR) and induce receptor creating loops for dimerization)
- Dimerization of catalytic site = obligatory step to relieve inhibitory constrains
- autophosporylation of intracellular domains on tyrosine residues –> enhance catalytic site, intracellular signaling
Following GF binding and phosphorylation of intracellular domains what forms?
SH2 domain which then binds GRB2 –>SOS = SH3 –> RAS/RAF
or
p85 –> p110 –> PI3k
to initiate intracellular signaling
What is the relationship between JAK STAT and cytokines?
JAK and resulting transcription factors they generate (STAT) provide intracellular signal transduction after an extracellular cytokine binds and initiates this pathway
Which TKI would be specific for M-phase inhibition?
- Polo like kinases and aurora kinase
- These are serine/threonine protein kinases involved with microtubule movement
- PLK1 localizes to centrosomes in early M - trigger for G2/M
What are cytokines?
- cytoplasmic tyrosine kinases recruited to cell surface molecules following receptor activation
- trigger intracellular events similar to RTKs
- transmit signals through JAK (janus kinase) = intracellular non-receptor kinase
Which signaling cytokine is immunosuppressive?
IL-10
(also IL-1Ra, IL-4, IL-11, IL-13, and TGFb)
Which phosphorylates threonine on CDK ?
CDK-activating kinase “CAK”
What regulates activated growth factor receptor signaling?
phosphatases –> dephosphorylate
List the most common phosphatases.
PTEN, protein tyrosine phiosphatases (PTP superfamily), protein serine/threonine phosphatases
- for every RTK there is a PTP associated that attenuates it
What is the function of phosphatase and TENsin AKA PTEN?
converts PIP3 to PIP2 via dephosphorylation
Anti-apoptotic proteins in the Bcl2 family?
Bcl-1, Bcl-xl, Bcl-w, Bcl2A1, Mcl-1
Pro-apoptotic proteins in the Bcl2 family?
Bax, Bcl-xs, Bak, Bad, Bik, Bim, Bid, Noxa, Puma
Which proteins does Tp53 over express to lead to apoptosis?
Bax, Noxa, Puma
Bcl-2 over expression leads to?
Bcl-2 - prolongs survival of cell and increases resistance to apoptosis
What are caspases?
cystein-dependent aspartate specific proteases
Caspases that initiate apoptosis?
CASP8, CAPS9, CASP10
Caspases involved in execution of apoptosis?
CASP3, CASP6, CASP7
What are the two apoptotic pathways?
1) Intrinsic AKA mitochondrial
2) Extrinsic AKA Death Receptor Pathway
Steps in intrinsic apoptosis pathway
1) Triggered intracellular stress signals ( e.g. DNA damage, oxidative stress, and growth factor withdrawal)
2) Release of pro-apoptotic proteins (Cytochrome C, SMAD, OMI) from mitochondria to cytoplasm
3) Cytochrome c release activates the apoptosome (multiprotein complex containing apoptotic protease-activating factor 1 (Apaf-1) and procaspase-9)
4 ) Caspase-9 activation –> caspase cascade (3/6/7) –> cell death
Steps in extrinsic apoptotic pathway?
1) Initiated by extracellular signals binding to death receptors on the cell surface (e.g. Fas (CD95), TNF receptor 1 (TNFR1), and TRAIL receptors)
2) Binding of ligands (e.g., Fas ligand, TNF-alpha) to death receptors leads to the formation of death-inducing signaling complex (DISC)
3) Activation of initiator caspase 8
4) Caspase cascade activation (3/6/7)
Overexpression of prosurival Bcl-2, Bcl-XL, MCl-1 results in?
Cancer
Where does dysfunction most commonly occur for the extrinsic pathway?
death receptor signaling or aberrant expression of anti-apoptotic proteins like FLIP (FLICE inhibitory protein) –> impaired apoptosis and cell survival
Where does dysfunction most commonly occur for the intrinsic pathway?
Overexpression of anti-apoptotic Bcl-2 proteins or loss of pro-apoptotic proteins –> inhibition of cytochrome c release –> suppression of apoptosis –> cell survival
How do the intrinsic and extrinsic apoptotic pathways cross talk?
caspase-8 activated by the extrinsic pathway can cleave and activate Bid, a pro-apoptotic Bcl-2 family protein, leading to mitochondrial outer membrane permeabilization and activation of the intrinsic pathway
List mechanisms to resist cell death.
loss of TP53 function, aberrant activation of anti-apoptotic proteins, inactivation of pro-apoptotic proteins, and deregulation of effectors responsible for implementing apoptotic and necroptotic signaling cascades
MOA of SMAC
AKA Diablo = pro-apoptotic by inhibiting IAP (inhibitor of apoptosis) –> promotes caspase 9
Activators of the extrinsic apoptotic pathway?
DR5 (TRAIL- death receptor 5), FAS, TNFR, granzyme via caspase 3 and 8 cleavage
How does Yunnan baiyao mediate apoptosis in vitro?
caspase mediated 3/7
Cellular features of apoptosis?
rounding of the cell, plasma membrane blebbing, cytoplasm shrinkage, alteration if plasma membrane symmetry, CONDENSED CHROMATIN, non-inflammatory
Which BH3 member causes necrosis and apoptosis?
Bid via caspase 8
MOA of thrombospondin 1?
binds CD36, block proliferation, activates FasL–> apoptosis
How does Bcl prevent apoptosis?
prevents release of cytochrome C from mitochondria
Why are carboplatin and gemcitabine synergistic? What results when given together?
Decreased DNA repair, induces cell cycle arrest and apoptosis, both inhibit NER
How does tp53 cause apoptosis?
Activates PUMA, NOX, Bak/Bim –> cyto C, caspase9, Apaf1 –> caspase cascade
Which requires energy apoptosis or necrosis?
Apoptosis; necrosis causes inflammation
Hallmark of cancer: evasion of apoptosis
upregulate Bcl-2, Bcl-XL, suppress Bax, Bim, Bak
Why are solid tumors hypoxic?
High consumption with compromised delivery systems leads to mismatch oxygen supply and demand
Intrinsic markers of hypoxia?
HIFa, GLUT1, CA-9, OPN
Which HIF (hypoxia inducible factors) is the master regulator most often in acute phase of hypoxia? Chronic?
- HIF1a acute
- HIF2 chronic
What is the primary pathway of tumor hypoxia? Steps in normoxia?
- Hypoxia inducible factor (HIF)
- HIF-α subunits are continuously synthesized but rapidly degraded via the ubiquitin-proteasome pathway.
- Prolyl hydroxylase domain proteins (PHDs) hydroxylate specific proline residues on the HIF-α subunits under normoxic conditions, targeting them for recognition by the von Hippel-Lindau tumor suppressor protein (VHL).
- VHL serves as the substrate recognition component of an E3 ubiquitin ligase complex, leading to polyubiquitination and subsequent proteasomal degradation of HIF-α subunits.
HIF pathway hypoxic conditions.
- oxygen-dependent hydroxylation of HIF-α subunits by PHDs is inhibited due to the decreased availability of oxygen.
- Stabilized HIF-α subunits translocate to the nucleus, where they heterodimerize with HIF-β/ARNT subunits.
- The HIF heterodimers bind to hypoxia-response elements (HREs) located in the promoters or enhancers of target genes, leading to their transcriptional activation.
- Promotes glycolysis and O2 delivery via angiogenesis
Target genes of HIF pathway under hypoxic conditions?
- glycolytic enzymes (GLUT1, LDHA)
- angiogenic factors (VEGF)
- erythropoietin (EPO)
- genes involved in pH regulation, iron metabolism, and cell survival.
Aberrant p53 function with HIF?
HIF1a = increased p53
HIF2a (chronic hypoxia) = decreased p53, growth advantage
Hypoxia mediated hallmarks of cancer?
- promotes VEGF/PDGF binding –> angiogenesis
- decreased apoptosis overtime –> hypoxia leads to RT resistance
- increased glycolysis
- stimulates metastasis
- promoting self renewal with cancer stem cells
- immune evasion: TAMS up regulate MMP7 –> less repsonseive to NK cells, increase PDL-1 expression, diverts dendritic cells, induces Treg
- genomic instability
How can HIF signaling promote metastasis?
Metastatic homing, transcription factors for epithelial to mesenchymal transition and increased cellular motility are hypoxia regulated
Hypoxia causes an acidic tumor microenvironment, which chemotherapeutics are affected by this?
- lower uptake of basic drugs - DOX
- higher uptake of acidic drugs - Chlorambucil
Which chemotherapeutics are affected by low drug exposure due to distance from blood supply in solid tumors with hypoxia?
- all but antracyclines and taxanes particulary affected
Chronic hypoxia can result in down regulation of mismatch repair. How does this effect chemotherapy?
- increased resistance to methylating agents
- increased sensitivity to platinum agents
Oxygen fixation hypothesis
oxygen interacts with secondary radicals on cellular molecules (DNA) formed by interaction with primary hydroxyl radicals produced by radiation effects on water in cells
Hypoxia causes radioresistance
Which 3 major proliferation pathways are activated via HIF up regulation? Activation? Tumorgenesis?
1) Notch Signaling
- activation HIF-mediated upregulation of Notch ligands such as Jagged and Delta-like proteins
- promoted cancer stem cell properties, epithelial-mesenchymal transition (EMT), angiogenesis, and metastasis.
2) PI3K/AKT/mTOR
- HIF-dependent upregulation of growth factors ( IGF-1) and direct inhibition of phosphatases
- promotes cancer cell survival, proliferation, angiogenesis, and resistance to therapy
3) MAPK/ERK
- HIF-mediated upregulation of growth factors ( EGF, FGF) and receptor tyrosine kinases (EGFR).
- cell proliferation, survival, invasion, and metastasis
How does Hif affect VEGF?
Anerobic conditions Hif is not ubiquinated by VHL so more VEGF is made
What are some mutations in HSA?
VHL(so increase Hif–>VEGF), bFGF, Ang1, p53, PTEN, PI3k/mTOR
- VHL-HIF in renal cell carcinoma people
What is the Warburg effect?
Cancer cells preferentially metabolize glucose to lactate via aerobic glycolysis regardless of the O2 status
What are the advantages of the Warburg effect?
- Rapid proliferation: Glycolysis provides intermediates necessary for the synthesis of macromolecules required for cell proliferation, such as nucleotides and lipids.
- Acidification of the tumor microenvironment: Lactate produced through aerobic glycolysis contributes to acidification of the extracellular environment, which promotes tumor invasion and metastasis while suppressing immune responses.
- Adaptation to hypoxia: Cancer cells in poorly vascularized regions of tumors can sustain energy production through glycolysis under hypoxic conditions.
- Saves energy
What are the key features of the Warburg effect?
- Increased glucose uptake via upregulation of GLUT1 (gluc into cells)
- Enhanced glycolysis
- Altered metabolism of pyruvate: pyruvate normally converted to acetyl-coA and enters the TCA cycle for oxidative phosphorylation. In cancer pyruvate to lactate by LDHA –> increases NAD+ for glycolysis
- Mitochondrial dysfunction: cancer cells often exhibit alterations in mitochondrial metabolism, such as reduced mitochondrial respiration and increased reliance on glycolysis for energy production.
Genes that induce angiogenesis?
VEGF-a, VHL
When does the angiogenesis switch occur?
When balance between angiogenic stimulators is favored over inhibitors
List the pro-angiogenic factors
VEGF-A, PIGF, FGF-3, FGF-4, IL-8, IL-6, TNF-a
List the anti-angiogenic factors
TSP-1, Endostatic, angiostatin, tumostatin, INF-a, PEX
What is the difference between angiogenesis and Neo-angiogenesis?
- Often used interchangeably
angiogenesis: - refers to the process of new blood vessel formation
- normal physiologic process involved in embyronic development and wounds
- in cancer refers to the formation of new blood vessels within the tumor microenvironment to support tumor growth, invasion, and metastasis
neo-angiogenesis:
- specifically emphasizes the newly formed vessels - refers to the process of forming new blood vessels within tumors, driven by angiogenic factors released by cancer cells and tumor-associated stromal cells
- hallmark of tumor progression and is associated with aggressive tumor behavior, increased metastatic potential, and resistance to therapy
Key triggers of angiogenesis and their pathways.
1) Hypoxia: HIF-1a stabilized acts as transcription factor to up regulate VEGF and angiopoietin2 (ANG2)
2) VEGF: up regulated with hypoxia, GF, cytokines –> stimulates endothelial cell proliferation, migration, and tube formation, and promotes the permeability of blood vessels
3) GF and cytokines: FGF, PDGF, TGFb, angiopoeitins, IL-6, IL8, PIGF
4) Extracellular matrix remodeling: degradation of the ECM by proteases such as matrix metalloproteinases (MMPs) facilitates the migration of endothelial cells and the formation of new blood vessels, also sends proangiogenic signals. CAFs producing VEGF
5) Inflammation: M2 (tumor promoting Mac) recruitment, bone marrow derived myeloid regulatory cells, TIE2- expressing macs
6) Mechanical forces: Shear stress and mechanical stretch hear stress can induce the release of endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO), which promotes endothelial cell survival and proliferation
What is the role of Tie-1?
VEGFR and Tie signaling coordinate process of angiogenesis, RTK expressed on blood vessels, involved in recruitment of pericytes and smooth muscle cell, maintain vasciular integrity
List the oncogenes when over expressed are pro-angiogenic
RAS, HER2, EGFR, SRC, MYC
Loss of function of these oncogenes results in angiogenesis
p53, PTEN, CDKN2A, VHL
Molecularly targeted agents designs to obliterate angiogenic pathways
Bevacizumab - VEGF
Ramucirumab - VEGFR2
Sunitinib, sorafanib, etc - VEGFR1-3
ALfibercept - VEGFR trap
Agents with antiangiogenic properties designed to block oncogenic pathways
Trastuzumab - HER2
Cetuximab - EGFR
Gefitinib, erlotnib - EGFR
Lapatinib - EGFR, HER2
Imatinib- ABL, PDGFRb, KIT
Agents with antiangiogenic activity originally developed for non-antiangiogenic indications
Metronomic chemotherapy, thalidomide
Genes related to invasion and metastasis
N-cadherin, SNAL, slug, TWIST, Zeb 1/2, MEK
How can you measure angiogenesis in a tumor?
micro vessel density - correlated with metastasis
List the steps of the metastatic cascade
- Invasion through basement membrane
- Intravasation
- Survival in circulation
- Extravasation
- Establishment of new growth
Increased SHH signaling causes?
proliferation, invasion, metastasis
also: embrogenesis, angiogenesis, and activation of CSCs
Metastasis suppressor genes?
MKK4, KISS1, NME, BrMS1. KAI1/CD82. BRMS1
Reduced cell adhesion of primary tumors in preparation for metastatic invasion involves?
TWIST, snail, slug, TGFb, ADAM10
- these are also involved in EMT
What is necessary for invasion of the metastatic cascade?
following change sin cell adhesion, matrix metalloproteases (MMPs) secretion which are enzymes that degrade the extracellular matrix
- also chemokines and cytoskeletal remodeling (RhoC)
Survival of circulating tumor cells (CTCs) is due tot he ability to resist what?
anoikis - apoptosis after the disruption of the interaction between epithelial cells and the ECM
- CTCs achieve this by traveling with platelets and aggregating to avoid NK cells
- platelets release EGF, VEGF, HGF, TGFb to help
What marker can be used to detect CTCs?
EpCAM = cell adhesion marker
- 2022 paper using collagen 1 and osteoclacin + cells via flow cytometry for OSA
What is meant by first pass capillary bed?
Tumors arrest in the first capillary bed they encounter typically - liver or lungs
CTC extravasation into new tissue and vascular remodeling is facilitated by?
MMPs, COX-2, EREG, ANGPT4
- potential drugable targets but limited success (e.g. MMPs)
Arrest of CTCs and early survival at distant sites is mediated by?
cell adhesion molecules (CAMS) specifically honing-CAM (h-CAM) aka CD44, chemokine and GF
-CD44 associated with poor prognosis in many human tumors AML, OSA, breast, etc
How does metastatic occur to bone?
CTCs express PTHrp, IL6, TNFa, to stimulate release of RNKL by osteoblast –> RNKL activates myeloid progenitor cells to differentiate into osteoclasts–> lytic activity release TGFb, insulin like GF, and BMPs to promote cell survival
Denosumab = moAB targeting RANKL to treat bone mets
What are selective pressure to metastasis? How does cancer overcome them?
1) Hypoxia: adapt to promote angiogenesis and glycolysis
2) Extracellular Matrix (ECM) Remodeling: secrete proteases (MMPs) to degrade the ECM and facilitate invasion
3) Immune Surveillance: evade immune surveillance through downregulation of major histocompatibility complex (MHC) molecules, expression of immune checkpoint molecules (e.g. PD-L1), and recruitment of immunosuppressive cells (e.g. regulatory T cells, myeloid-derived suppressor cells).
4) Anoikis Resistance: binding to platelets, aggregating
5) Chemoresistance and radioresistance: various mechanisms
6) Adaptation to Distant Microenvironments: interaction stromal cells, remodeling of the ECM, and metabolic reprogramming
7) Competitive Fitness: metastasis is inefficient and most CTCs do not survive without the ability to evade selective pressure
What is the benefit of epithelial to mesenchymal transition (EMT)?
- epithelial loss of apicabasal polarity and cell to cell contact with increase cell motility and invasion similar to mesenchymal cells
- develop ability to undergo invasion, migration, and intravasation for metastasis
Transcription factors primarily involved in EMT?
Twist, snail, slug
- zen through miRNA
Markers of epithelial-like tumor cells?
cell adhesion proteins: E-cadherins, Zo-1, laminin, desmoplakin, occludin
Markers of mesenchymal-like tumor cells
cytoskeletal proteins: N-cadherin, vimentin, B-catenin, a-SMA
List EMT activation factors
HGF, EGF, PDGF, IL6/JAK/STAT3, TGFb, WNT/B-catenin, MMPs, microRNAs
What markers are associated with EMT in feline oral SCC?
P-cadherin, Twist, HIF-1α, and PDLI = highly expressed
E-cadherin = co-expressed
Vet Path 2019
When is mesenchymal to epithelial transition (MET) thought to be utilized?
- mesenchymal cells or epithelial cells MET as the final step in EMT after the activation, development or an invasive phenotype, and colonization at a distant site occurs, cell will revert to original epithelial phenotype
- unclear benefit, happens during embryonic development
- inducing MET could improve TX outcomes as epithelial cells are considered more sensitive to chemo than mesenchymal
pH of normal cell? Tumor cells?
norm >7.4
tumor 7.2
- develops due to necrotic/hypoxic areas in the tumor
What results in acidosis of the TME?
aerobic glycolysis with accumulation of lactate, hydrolysis of ATP, glutaminolysis, and CO2 production
TME may be as low as 6.5 pH esp in regions away from blood vessels
Extracellular TME acidification leads to…
- local tumor invasion and metastasis
- protease activation –> remodeling of cell matrix and cell-cell interactions + intracellular tumor basic Ph –> migration through ECM
How does the acidity of the ECM inhibit immune responses?
- accumulation of H+ and lactate interfere with T-cell activation and release of cytokines
- acidity within tumors also contributes to drug resistance by reducing cellular uptake of many basic drugs
Summarize how hypoxia and aerobic glycolysis drive cellular migration?
- Stimulates HIFa –> expression of genes encoding cytokines, chemokines, growth factors, and extracellular matrix (ECM) remodeling enzymes –> directional migration of cells towards areas of lower O2 tensions
- hypoxia-induced secretion of VEGF and stromal-derived factor 1 (SDF-1) can act as chemoattractants for cancer cells, guiding their migration toward blood vessels
- High rates of glycolysis generate ATP rapidly to fuel cytoskeletal rearrangements, cell membrane dynamics, and cell protrusions essential for migration
- Enhanced glycolytic flux supports the migratory phenotype by providing ATP and metabolic intermediates for biosynthesis, maintaining redox balance, and regulating signaling pathways involved in migration and invasion
- modulate the composition and stiffness of the ECM, influencing cellular adhesion, motility, and migration + increased secretion of ECM-degrading enzymes such as matrix metalloproteinases (MMPs), facilitates invasion and migration of cancer cells through the ECM
How does hypoxia of the TME influence tumor immunity?
- promotes an immunosuppressive and immune tolerant environment
TAMS in hypoxic TME and outcome?
- tumor associated Macs (TAMS) recruited via CSF1 and VEGF undergo conversion to M2 (immunosuppressive Macs) via IL4 and IL10
- M2 Macs up regulate MMP7 –> tumor less responsive to NK and T cells
Myeloid derived suppressor cells (MDSC) in hypoxic TME and outcome?
- MDCS accumulate in response to GM-CSF, VEGF, and prostaglandins in TME
- induce T cell anergy
-HIF1a produces arginine and nitric oxide by MDCS –> further immunosuppression via PDLI upreguation on MDCS and T cell tolerance
Hypoxia effects on dendritic cells?
Diverts dendritic cells responsible for Ag presentation and activation of TCell further leasing to immuno suppression
Where is VEGFR expressed and what does it do?
Vascular endothelium, Drive migration and proliferation
How does inflammation in tumors influence cell migrations?
- Recruitment of Immune Cells: immune cells release cytokines, chemokines, and growth factors that promote cancer cell migration and invasion.
- E.g. TAMS secrete TNF-α, IL-6, and TGF-β –> increased cell migration/invasion & EMT
- Induction of Angiogenesis: VEGF, IL8, FGF –> endothelial cell migration, tube formation, angiogenesis
- ECM Remodeling: MMPs and capthesins degrade ECM proteins and facilitate cancer cell migration
- Chemotactic gradients in areas of tissue damage
- Immune Cell-Induced EMT: cytokines such as TGF-β, TNF-α, and IL-6 can activate EMT-inducing transcription factors (e.g., Snail, Slug, Twist)
- Immune Cell Trafficking: neuts and monos can promote seeding and colonization of pre metastatic niche
What are the 4 classes of proteinases?
Serine, cysteine, aspartic, and metallproeinases
- aberrantly expressed in TME
- normally involved in digestion, protein turn over, wound healing, etc
Catalytic type proteinase Cystein association with malignancy, inhibitors, substrates?
malignancy: cathespins B, L, H and calpains
inhibitors: kinogens, Kalpastatin
substrate: ECM, focal adhesion proteins, cell signaling proteins
Catalytic type proteinase Aspartic association with malignancy, inhibitors, substrates?
malignancy: cathepsin D
inhibitors: not known
substrate: ECM
Catalytic type proteinase metallo association with malignancy, inhibitors, substrates?
malignancy: MMPs 2,3,7,9,13,14
inhibitors: TIMPS (tissue inhibitor of MMPs)
substrate: ECM, GF, cytokines
Catalytic type proteinase Serine association with malignancy, inhibitors, substrates?
malignancy: uPA, tPA (plasmagin activators)
inhibitors: PAI (plasming activator inhibitor)
substrate: plasmagin, latent MMPs
Which factors in the TME contribute to the immunosuppressive barrier of tumors?
Tregs, MDSCs, mesenchymal stem cells (MSC), TGFb, immunoglobulins
What IL connects innate & adaptive immunity?
IL12
What do TLR do?
Recognize microbial products, 1st step innate immune system
How do liposomal-DNA complexes work?
Activates innate immune system by CpG-oligonucleotides; Induce NK and release IFNy
What is the primary immunosurveillance molecule?
IFNγ-levels should change if there has been an immune response
What are the 3 phases of the immune response to tumor formation or imnoediting hypothesis?
1) elimination - removal of immunogenic tumor cells by the immune system (less immunogenic cells survive)
2) equilibrium - tumor growth and immune destruction are equal = dormancy
3) escape - tumor growth ensues due to decreased immunogenicity, immune suppression and rapid tumor cell growth
What cells express MHC-I? MHC-II?
MHC-I-all cells MHC-II-only APC
Is MHC-I use endogenous or exogenous peptide pathway?
MHC-I uses endogenous so intracellular contents
How are TH stimulated/activated?
APC-MHCII-activates Th2(by IL4)–>IL4, activates CD4-make Ab and switching, IL2- more CD8;
APC MHCI-activates CTL, and Th1(by IL12)–>CD8, IL2, GMCSF, IFNy
How does peripheral tolerance occur?
Escaped central tolerance; Deletion of clonal Tcells(lack of costimulatory), Anergy (lack of costim), Ignorance, Regulation(Treg)
What are the positive markers/cell types for Treg?
CD4, CD25, FoxP3
What do Treg secrete?
TGFb, IL10
How do NK cells recognize TAA or become activated?
Primary-recognize decreased MHCI on cells; 2nd stress signal MIC activates NK on cells
How do tumors evade the immune system?
Decreased MHC-I expression (CD8 miss), Immunosuppressive cytokines (TGFb, IL10, TNFa), Increased Treg, Increase Myeloid derived suppressor cells, Fail to express costimulators, DC dysfunction, Direct death through FasL on tumor
What evidence is there that the immune system is involved with tumors?
Spontaneous remission w/o tx; Paraneoplastic autoimmunity; CTL in tumor; Increased risk of tumors in immunosuppressed
How can you tell a vaccine is causing immunomodulation?
Delayed type hypersensitivity & lymphocyte proliferation assays
Check point molecules are expressed by which cells?
T-Cells
Co-inhibitory = PD-1, CTLA04, TIM03, VISTA, BTLA, B7-H3, B7-H4, Lag3
Co-stimulatory = CD28, OX40, GITR, ICOS, CD137, CD27, CD40L, CD122
- gilvetman inhbiits PD1 on T cell
PDL-1 is expressed by?
myeloid cells (monos, Mac, dendritic cells) and tumor cells
How do tumors become invisible?
Down regulation of MHC class 1 & expression of immunosuppressive cytokines
What are allogenic vax?
TAA from same species but not same animal/person, Can be massed produced but may not have correct TAA that are on patients tumor
How did the combo of toceranib+cyclophosphamide effect the immune system in cancer dogs?
↓Treg (mostly by day 14) and ↑IFNy (6wk combo tx, when Treg lowest)
T helper cells (CD4) recognize peptides presented on MHC class II of APCs. After activation through TCR rearrangement what are the 4 pathways of Th0 differentiation and effector rearrangement?
1) IL-12 –> Th1 –> IFNy, TNFa, IL-2 –> cytotoxic lymph function tumor/vrius/intracellular bacteria
2) IL-4 –> Th2 –> IL-4, IL-5, IL-6, IL-10 –> B-cell, allergies, asthma, IgE
3) TGFb, IL-6 –> Th17 –> IL-17 –> autoimmunity, tissue inflammation
4) TGFb, IL-10 –> Treg (FOXP3) –> TGFb, IL-10 –> suppression of immunity
What is the role of tregs?
- natural CD4+CD25+ T cells for self tolerance
- if removed mice die of lymphoporlifertative disease
- suppress other T cell responses
- increase in cancer Tregs inhibit the activation and effector functions of cytotoxic T cells and NK cells, limiting their ability to eradicate tumor cells.
- Use IL10 and TGFB to resolve inflammation
- potential therapy to limit graft vs host disease with BMT
Immunosuppressive TILS?
Tregs, MDSC, M2 TAMS
What are the subsets of myeloid derived suppressor cells?
- Granulocytic: touch other cells to suppress them via ROS
- Monocytic: not Ag specific, no cell to cell contact, use arganise and NO to suppress other cells
- both found in TME and PATHOGENIC (not normal cells)
- GR1+ (neut marker), CD11b+ w/o other Mac or DC markers
Which tumor infiltrating lymphocytes have been assocaited with a better prognosis?
- CD3+, CD8+
- cytotoxic T cell and NK cells aid in tumor immunosurrveilance
- better response to immunotherapy when present
- M1 TAMS
Role of M1 TAMs
- classical
- pro inflammatory via IL-12, TNFa, IL-7b, IL-6a for host immune response
- antimicrobial activated by LPS
- kill tumors produce cytotoxic molecules NO and reactive oxygen species ROS to induce apoptosis and necrosis i
- activate CTLS and NKs via IL-2 and TH1
- Promote TH1 immune response via IFN-γ = cell mediated
- tissue remodling and repair
Role of M2 TAMS
- anti parasitic
- TH2 immune erpsonse
- promote tumor growth, angiogenesis and mets via VEGF, TGFb
- Suppress CTLS and NK cells
- IL4 and IL14 antifilammatory
What are the 5 types of TAAs (tumor associated antigens) and relevant examples?
- Mutation Ag
- BRAF
- driver/passenger mutations in AA –> mutated protein expression on MHC –> unique neoAg rec as foreign Tcell response –> antitumor immunity - Cancer - testis Ag
- MAGE, BAGE, GAGE, NY-ESO-1
- only expressed by tumor or normal placental germ cell tissue - Differentiation Ag
- tyrosinase in melanoma
- expressed on normal tissue too - Viral AG
- Epstien Barr, HPV - AG of unique postranslation modification
- could b ID by CAR Tcells
