Lecture 6. tRNA & Protein Synthesis in Cancer Flashcards
Describe how Ribosome Production is Consistently Elevated in Cancer
Ribosomes responsible for protein synthesis – assembled in nucleoli, specialised regions of nucleus which assemble around sites of large ribosomal RNA genes
Genes transcribed at very high rates in every type of cancer by RNA pol I
Ribosomal proteins assemble on the nascent transcript such that by the time the RNA polymerase reaches the end of the rRNA gene much of the ribosome has already been assembled
The nucleoli are areas of dense material – high intense activity
Pathologists determined enlarged nucleoli often a sign of cancer
In many cases size of nucleoli can provide prognostic information
Describe how MYC Stimulates Transcription by Pols I & III
Ribosomes contain 4 rRNA molecules, three come from a pre-rRNA precursor that is made by pol I, whereas the other (5S rRNA) is made by pol III
MYC directly stimulates pre-rRNA synthesis by pol I & 5S rRNA synthesis by pol III
Shown using a MycER inducible system in which MYC is fused to the ligand-binding domain of the oestrogen receptor and can be activated with the ligand tamoxifen
MYC also stimulates tRNA synthesis by pol III
tRNA & 5S rRNA gene promoters do not contain DNA sequences that are recognised by MYC
MYC-ER construct – MYC protein was fused to the ligand-binding domain of the oestrogen receptor, could be controlled by synthetic oestrogen
In the absence of the ligand the construct remains in the cytoplasm
Tamoxifen used as ligand, enters nuclei and attaches to target genes
Rapid induction of large ribosomal RNA –precursor of 3 rRNA subunits and 5s rRNA, within 3 hours
Genes that code for large rRNA contain e boxes which are directly recognised by MYC
These sequences weren’t apparent in 5s rRNA or tRNA genes but these were still induced MYC
Describe how MYC is Recruited to tRNA & 5S rRNA Genes by TFIIIB
MYC is recruited to tRNA & 5S rRNA genes by protein/protein interactions with pol III-specific transcription factor TFIIIB
MYC recruits HATs that stimulate txn by pol III
Presence of MYC at large proportion of tRNA genes
Lack of DNA sequence recognized by MYC – due to protein-protein interaction instead
MYC recruits HATs which in turn acetylate local nucleosomes, leads to pol III transcription of tRNAs and 5s genes
Describe how it was experimentally determined that Certain tRNAs Promote Metastasis
To determine if the level of a molecule influences behaviour, test effect of specifically manipulating (increasing &/or decreasing) level of that molecule
To test if tRNAGlu-UUC overexpression contributes to metastasis:-
1. A tRNAGlu-UUC overexpression vector was introduced into breast cancer cells
- this increased metastatic spread to lungs of mice
2. When tRNAGlu-UUC was depleted from highly metastatic breast cancer cells by RNAi, metastatic spread decreased
Therefore, breast cancer metastasis responds to levels of tRNAGlu-UUC
To test the significance of levels of this tRNA for breast tumourigenesis – tested what happened if level was specifically raised or lowered
Look for changes in behaviour
Took an expression vector carrying the tRNA and transfected it into a breast cancer cell, chosen because relatively stationary- not prone to high levels of metastasis
Marked the cells with luciferase reporter gene driven from constitutive promoter
Transplanted cells into mice
Monitored bioluminescence in vivo without having to interfere with mice
Control cells were just transfected with empty vector not containing a tRNA
Location remains close to site where they were introduced
However cells with overexpressed tRNA spread considerably and also increased in number
Different colours indicate different intensities of bioluminescence
Used RNA interference to deplete the tRNA – when these cells transplanted those with depleted tRNA showed much lower levels of bioluminescence
Implicated tRNA as having oncogenic role
Describe how Elevated tRNAGlu-UUC Increases Metastasis by Selectively Altering Protein Synthesis
two different tRNAs add Glu to growing polypeptides – tRNAGlu-UUC & tRNAGlu-CUC - decode GAA & GAG codons, respectively
tRNAGlu-UUC overexpression was found to selectively increase translation of mRNAs with multiple GAA codons
some of these GAA-rich mRNAs encode proteins that stimulate metastasis
changes in protein expression were revealed by proteomic analysis
tRNAs are decoders – translate genetic code into amino acids
This tRNA affects protein synthesis in some way
Two different codons code for glutamic acid GAA and GAG
Translated by two different tRNAs – synonymous, give same AA
Overexpression of tRNA with UUC anticodon
Explain how Alcohol Stimulates tRNA Expression & Cancer
Excessive alcohol consumption is a risk factor in several cancer types, esp. oesophagus & breast
E.g. 1 unit/day of alcohol increases breast cancer risk by 7% - risk increases with higher intake
Ethanol treatment of breast cancer cells stimulates tRNA expression strongly
Oestrogen (E2) & ethanol (EtOH) synergize to stimulate tRNA expression & transform breast cells
Describe how Oestrogen Stimulates Expression of tRNA, rRNA & Ribosomal Proteins
Oestrogen is strongly implicated in the aetiology of breast cancer
Oestrogen stimulates growth & proliferation of breast cells
Transcription of genes encoding rRNA, tRNA & proteins involved in translation is strongly induced in breast cells by synthetic oestrogen E2
Effects can be countered by anti-oestrogens such as tamoxifen
Adipose provides oestrogen in post-menopausal women & obesity increases risk of breast cancer by ~30%
Describe how mTOR Stimulates tRNA Synthesis & Translation in Response to Growth Factors
Synthesis of tRNA is restrained by RB & a pol III-binding repressor called Maf1
Maf1 is inactivated by mTOR-mediated phosphorylation
mTOR also phosphorylates & inactivates 4E-BP, a repressor of translation initiation
mTOR is activated by the PI3K pathway in response to growth factors e.g. insulin
PTEN is a tumour suppressor that inhibits PI3K signalling by dephopsphorylating phosphoinositides
In many cancers, mTOR is hyperactive due to activation of the PI3K pathway through mutations in Akt, PI3K, PTEN, Ras or growth factor receptors
TFIIIB is repressed by RB protein to inhibit tRNA synthesis
Can also be inhibited by Maf1 protein which inhibits RNA polymerase itself
Maf1 turned off by mTOR protein kinase at end of PI3K kinase pathway, downstream of Ras and growth factor receptors
4E-BP is another target of mTOR – translation repressor binds to EIF4 cap binding factor responsible for recruiting mRNAs to ribosomes
When unphosphorylated 4EBP inhibits translation initiation
mTOR can stimulate translation by phosphorylating and inactivating this translational repressor
mTOR can stimulate both protein synthesis and tRNA synthesis and thereby have profound effect on accumulation of biomass and cell growth
PTEN deleted/inactivated in many cancers
In other cancers you get mutational activation of Ras, GF receptors etc
Elevated levels of signalling through this pathway
Describe how mTOR Inhibitors are Used in the Clinic
Several mTOR inhibitors have been developed & tested in clinical trials
Rapamycin derivatives (rapalogs) such as Afinitor (everolimus) have been approved for treatment of advanced renal cell cancer
Proved less successful in the clinic than had been predicted from mouse models
One problem is that a negative feedback loop exists between mTOR & the PI3K pathway – by preventing this feedback, mTOR inhibitors stimulate Akt signaling, which increases survival of cancer cells
Inhibition of mTOR sometimes causes activation of MAPK signalling to ERK - a solution may be to combine mTOR inhibitor with MAPK pathway inhibitor
Describe how Obesity Raises PI3K & mTOR Activity & is a Major Cancer Risk
Excess weight or adiposity increases incidence & severity of many cancers
Mechanisms are complex & vary, but a major cause is increased systemic insulin levels, which activate PI3K signalling & mTOR
One of the principle mechanisms is excessive signalling through PI3K pathway to mTOR – elevated in obese patients due to higher levels of systemic insulin
Explain Re-purposing Metformin for Cancer Therapy
Metformin is a safe, cheap & effective drug widely used to treat insulin resistance in type 2 diabetes, esp amongst people who are overweight
Metformin reduces circulating levels of insulin & glucose by inhibiting hepatic gluconeogenesis
Also inhibits the mitochondrial respiratory chain, causing energy deficit within cells
AMP kinase regulates mTOR in response to energy status (AMP/ATP ratio) – suppresses mTOR activity when metformin causes energy deficit (elevated AMP/ATP)
Epidemiology shows that diabetics receiving metformin have lower cancer incidence & cancer death
Clinical trials with non-diabetic cancer patients showed mTOR inactivation & reduced cancer cell proliferation in breast & colorectal cancers
No impact on survival in a clinical trial of advanced pancreatic cancer, but ongoing large clinical trials are monitoring effects of metformin on survival & recurrence in several cancer types
Potential for re-purposing metformin as a cancer therapy
Metformin cost per month (2019) is £3.41 - Afinitor (everolimus) £2,473 per month (2017)
Obesity also frequently associated with type 2 diabetes -treated with drug metformin
