Lecture 4 - The RB pathway Flashcards
What is RB?
RB was discovered as the tumour suppressor inactivated in retinoblastoma, a rare cancer of the retina
Inheritance of one inactive Rb allele gives a ~90% chance of retinoblastoma by 6yrs of age, after somatic mutn of second allele
~95% cure rate, but survivors are predisposed to sarcomas later in life, after loss of second Rb allele
Somatic mutn of both Rb alleles is common in many tumour types e.g. almost all small cell lung cancers
Describe the structure of the RB protein
RB protein contains a central domain called the pocket that interacts with many proteins
Several DNA tumour viruses encode oncogenic proteins that neutralise RB by binding a cleft in the pocket e.g. E7 protein of human papillomavirus (HPV)
Cervical cancers usually have WT Rb genes, but the protein is neutralised by E7
An N-terminal domain of RB resembles the pocket
much of the rest of RB is unstructured & flexible
C-terminal region is bound by protein phosphatase 1 (PP1) & cyclin-dep kinases cdk2 & cdk4
Pocket targeted by several DNA tumour viruses eg HPV
Rare to find RB mutations in cervical cancer because HPV inactivates RB – no selective pressure for RB mutation
Describe how RB controls the cell cycle
RB is unphosphorylated & active in G1 phase
late in G1, CDK4 (or CDK6) bound to cyclin D phosphorylate RB – this commits cells to leave G1 & progress through cell cycle – referred to as the “Restriction point”
Cyclin D is expressed in response to mitogens - RB therefore couples cell cycle entry to mitogenic signals – “Gatekeeper of the cell cycle”
in S phase, cdk2/cyclin E phosphorylates additional sites in RB
Phosphorylation is reversed late in M phase by protein phosphatase 1 (PP1)
Phosphorylation causes conformational rearrangement of RB
As long as RB is unphosphorylated, cells stay in G1 or become quiescent (G0) or senescent
RB blocks passage of cells from G1 into S phase when unphosphorylated
RB phosphorylated by cdk4/6 when complexed with cyclin D – allowing cells to progress
Changes in phosphorylation causes conformational change affecting interaction with partners
Describe how RB phosphorylation is deregulated in cancers
Although RB is mutated in some cancers (e.g. retinoblastoma), most cancers retain wild-type RB
Some cancers neutralise RB with oncogenic proteins that bind pocket domain (e.g. cervical cancer)
often RB phosphorylation is deregulated
E.g. many breast cancers overexpress cyclin D causing inappropriate RB phosphn
INK4 tumour suppressor genes encode proteins p16 & p15 that bind CDK4 & CDK6 to prevent cyclin D binding
INK4 genes are deleted in many cancers (e.g. melanoma), deregulating RB phosphn
compromised RB function allows cancer cells to evade growth suppression (hallmark)
Although RB is mutated in some cancers (e.g. retinoblastoma), most cancers retain wild-type RB
Some cancers neutralise RB with oncogenic proteins that bind pocket domain (e.g. cervical cancer)
often RB phosphorylation is deregulated
E.g. many breast cancers overexpress cyclin D causing inappropriate RB phosphn
INK4 tumour suppressor genes encode proteins p16 & p15 that bind CDK4 & CDK6 to prevent cyclin D binding
INK4 genes are deleted in many cancers (e.g. melanoma), deregulating RB phosphn
compromised RB function allows cancer cells to evade growth suppression (hallmark)
All of these molecular pathways have the same effect of releasing cancer cells from an important growth suppressing mechanism – hallmark of cancer
Describe how CDK4/6 Inhibitors are Used to Treat Breast Cancer
Palbociclib is one of several inhibitors of CDK4 & CDK6 that is approved for treatment of breast cancer, where cyclin D is often overexpressed
Elicits reversible cell cycle arrest & extends life by 10 months on average
Most patients suffer neutropenia (depletion of neutrophils) & increased susceptibility to infection – anemia also common
Cancers eventually become resistant to palbociclib, sometimes due to decreased expression or mutation of RB
Describe how RB Binds & Inhibits E2F
Best characterized RB targets are E2F txn factors, which bind & regulate genes involved in DNA replication & cell cycle progression e.g. cyclin E gene
E2F binds promoters of target genes & then uses its txn actn domain to recruit TFIID
TFIID recruitment is rqrd for txn of protein-coding genes, but is inefficient – assistance by txn activators such as E2F accelerates a rate-limiting step
RB pocket binds & masks txn actn domain of E2F
Phosphn of RB causes release from E2F & allows txn of E2F target genes
RB interacts with variety of other proteins, mainly TFs
TFs promote cell cycle expression and proliferation by stimulating expression of genes that promote DNA replication
TFIID TBP containing txn factor that binds to TATA boxes in promoters
Txn activation domain of E2F interacts with TFIID and this helps recruit TFIID to its target genes
P53 also activates transcription in this way
TFIID recruitment often rate limiting for txn initation so anything that accelerates recruitment of TFIID is likely in impact upon gene transcription
RB counteracts this by binding to txn activation domain of E2F so it can only interact with TFIID – but only in unphosphorylated form
Describe how RB Recruits HDACs & DNMT1
When bound to E2F, RB recruits histone deacetylases (HDACs) & DNA methyltransferase (DNMT) 1 to repress E2F target genes
DNA methylation occurs at CpG sites, that are common at mammalian promoters
CpG methylation blocks binding by some TFs e.g. MLL1 histone methyltransferase that marks active promoters with H3K4me3
histone deacetylation reduces accessibility of DNA & removes marks that recruit TFs with bromodomains e.g. TFIID
RB is not DNA-binding factor – recruited via other proteins txn factors such as E2F
When tethered to E2F it can not only prevent the induction of transcription via E2F txn activation domain but it can also actively promote silencing of the genes by recruiting histone deacetylases which remove the acetyl groups from histones thereby promoting the interaction of the histones with DNA so as to reduce accessibility
Also remove acetylated lysine signals that help recruit factors with bromodomains such as the TAF1 subunit of TFIID
In addition RB recruits DNMT1 which methylates DNA on CpG sites
Methylated CpG is resistant to recruitment by various TFs such as MLL1
Explain how RB & p53 Pathways are Usually Both Deregulated in Cancers
As E2F activates the ARF promoter, loss of RB function triggers induction of ARF & hence p53 – “oncogenic stress”
Progression of tumours after RB inactn therefore requires p53 inactn
Deletion of INK4/ARF locus deregulates both pathways & is common in many tumour types, e.g. melanoma
MDM2 binds & inactivates both RB & p53
Mutns in p53 or RB are rare in cervical cancer – HPV encodes E6 protein that targets p53 for ubiquitination & degrdn & HPV E7 protein that binds & neutralises RB
ARF promoter also has binding sites for E2F – important because controls induction of p53 in response to oncogenic stress
When RB is active unphosphorylated and bound to E2F it’s suppressing expression of ARF promoter
If something happens that inactivates RB such as DNA tumour virus or hyperphosphorylation of RB, ARF promoter is derepressed, ARF gets induced, it then inactivates MDM2 allowing induction of p53 because it’s no longer getting degraded
So you get a p53 response such as cell cycle arrest or apoptosis in response to that oncogenic signal that had inactivated RB
For this reason for tumours to progress after inactivating RB they generally need to also inactivate p53
Eg through deletion of INK4A/ARF locus
Overexpression of MDM2
MDM2 and HPV can take out both key tumour suppressors
Describe how RB Binds & Regulates Many Transcription Factors
As well as E2F, many other txn factors are bound & regulated by RB
E.g. UBF which recruits pol I to rRNA genes
E.g. TFIIIB, which recruits pol III to tRNA genes
By binding UBF & TFIIIB, RB can suppress production of rRNA & tRNA – this limits protein synthesis & hence growth
Phosphorylation of RB causes release of UBF & TFIIIB, restoring prodn of rRNA & tRNA, protein synthesis & growth
rRNA & tRNA are overexpressed in cancers
UBF important for synthesis of rRNA by RNA polymerase I – very important because rRNA most abundant form of RNA in cells so having restraints upon rRNA synthesis can impact strongly upon ribsome biogenesis
Regulating tRNA synthesis impacts protein synthesis - growth inhibitory effect
RB can suppress protein synthesis by limiting production of both rRNA and tRNA
When RB is phosphorylated it loses these interactions with TFs and releases them allowing cells to grow again
Describe how Phosphorylated RB Binds & Regulates p65
Most RB targets are released when RB is phosphorylated e.g. E2F, UBF, TFIIIB
Txn factor p65 (RelA) is bound by phosphorylated RB & released when RB is dephosphorylated
p65 is one of five members of the NF-κB/Rel family, each with a Rel homology region (RHR) – p65 may be the only family member that binds specifically to RB
RHR mediates dimerization & DNA binding between NF-κB/Rel family members - different homo- & heterodimers bind distinct DNA seqs – substantial combinatorial diversity
RHR is N-terminal domain responsible for dimerization and DNA binding
Can get both heterodimers and homodimers that differ in their specificity – provide combinatorial diversity
Explain how RB May Suppress Cancer Immunity By Inhibiting p65
NF-κB/Rel family members induce genes encoding many components of immune system, e.g. cytokines & PD-L1 (programmed cell death ligand 1)
PD-L1 contributes to immune tolerance, protecting against tissue attack & auto-immunity
Immune cells, including T, B & NK cells, express PD-1 cell surface receptor that prevents activation of immune response when engaged by its ligand PD-L1
Tumour cells can evade immune destruction by inducing PD-L1
Tumour cells induce overexpression of PD-L1 to protect themselves from immune attack
Tumour cells commonly express abnormal antigens which are recognised by our immune systems as foreign leading to attack and destruction of tumours
Overexpression of PD-L1 allows them to evade immune system
PD-L1 is induced by p65
RB inhibits this induction and therefore provides protection against immune evasion
What is Immune Checkpoint Blockade Therapy?
Monoclonal antibodies that bind PD-L1 or PD-1 are proving to be effective for treating patients with metastatic disease
Prevent PD-L1 from engaging PD-1 to suppress immune attack
For example, Keytruda is a monoclonal antibody against PD-1 that is used to treat several cancer types, including metastatic lung cancers – extends life by ~16 months relative to standard chemotherapy
Expensive – 2019 list price is £5,260 for each three-weekly infusion, although NHS has negotiated an undisclosed discount
Pd1 protein that can be effectively targeted
Specifically targeted very precisely due to specificity of antibodies
Antibodies block interaction between ligand and receptor and that prevents evasion of immune system
