RNA variants and their possible
role in therapeutic resistance
Benoit Chabot
Laboratory of Functional Genomics of the Université de Sherbrooke
Département de microbiologie et d’infectiologie
Faculté de médecine et des sciences de la santé
Sherbrooke, Québec
Canada
Potential Conflict of Interest
• Dr. Benoit Chabot
– None
Cumulative mutations
give cancer
• Activating oncogenes
• Inactiving tumor
suppressors
• More or less of it
(promoter,
multiple copies)
93% of our 25 000 genes are
alternatively spliced
Combinatorial mode of splicing control
makes the process highly vulnerable to mutations
From Chen and Manley 2009 Nature Reviews Molecular Cell Biology 10, 741-754
Combinatorial mode of splicing control
makes the process highly vulnerable to mutations
ANTI - APOPTOTIC (upregulated in cancer cells and
confers resistance to many anticancer drugs)
1
Bcl-xL
2
Distal Prox.
5’ss 5’ss
ATG
1
Bcl-xS
2
1
2.1
3
3’ss
Stop
2.1
2
PRO - APOPTOTIC
3
3
Combinatorial mode of splicing control
makes the process highly vulnerable to mutations
P
SRp30c
xS
X
xL
ISE
SAP
155
F/H
P
A1
Sam68
Fyn
Impact of mutations in exons
(coding sequences)
• Nonsense mutations
GGAAGGGA
Impact of mutations in exons
(coding sequences)
• Nonsense mutations
GGuAGGGA
Impact of mutations in exons
(coding sequences)
• Nonsense mutations
GGuAGGGA
BRCA1
NF1
Impact of mutations in exons
(coding sequences)
• missense mutations
Gly
Gly
Arg
GGAGGAAGG
Impact of mutations in exons
(coding sequences)
• missense mutations
Gly
Ala
Arg
GGAGcAAGG
Impact of mutations in exons
(coding sequences)
• missense mutations
GGAGcAAGG
Impact of mutations in exons
(coding sequences)
• silent mutations
Gly Gly Arg
GGAGGGAGG
Impact of mutations in exons
(coding sequences)
• silent mutations
Gly Gly Arg
GGAGGcAGG
Impact of mutations in exons
(coding sequences)
• synonymous mutations
GGAGGcAGG
Synonymous mutations in human CFTR exon 12 induce exon skipping.
86% of the positions affect alternative splicing,
only a fraction affects the protein coding capacity
Pagani F et al. PNAS 2005;102:6368-6372
©2005 by National Academy of Sciences
Genetic polymorphisms (SNPs) and splicing
 10% of alternative cassette
exons show splicing differences
between individuals
 Differences linked to
proximal SNPs
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Thus, genetic polymorphisms
can have a dramatic
impact on splicing profiles
Hull et al. 2007. PLoS Genetics
Another way to alter splicing is by changing
the expression of the splicing regulators
+
fox2
fox2
At least 100 cases of
alternative splicing
changes specific to breast
and ovarian cancers are
caused by a drop in the
expression of Fox2
Venables et al. 2009 NSMB
Modulating the relative levels of splice variants
2 siRNAs
siRNA
siRNA
Ovarian and Breast Cancer Associated Changes in
Splicing Influence the AKT-Dependent Pro-Survival
Pathway
Cytokines/TNF
FN1
MAP3K7 (TAK)
SYK
PI3K
Ras
MINK
MAPK
AKT
GSK3b
MCL-1
cell survival
(Prinos et al. in revision)
Relevance to drug resistance ?
Mechanisms of drug resistance
Gottesman 2002 Ann. Rev. Med 53:615-627
Drug transport
• Uptake
SLC superfamily 360 genes (involved in resistance to
methotrexate, gemcitabine, doxorubicin, platinium
agents, paclitaxel, possibly more)
Some evidence that genetic polymorphisms in these
genes affect pharmacokinetics
• Efflux
MDR1, other ABC members (48 genes) and other
transporters
Alternative splicing in drug transporters
Drug metabolism
• Cytochrome P450 (57 genes)
• Glutathione-S-transferase, etc …
Genetic polymorphisms in these genes
are associated with intrinsic resistance
What are the chances that this
involves differences
in splicing control ?
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DNA damage response
• Goal of anticancer treatment is to create enough
damage to have cells go into apoptosis, senescence
or necrosis
• Sensors of DNA breaks (H2AX, ATM, ATR, CHK2)
• DNA repair machinery
• Deficiencies in DNA repair associated with resistance
to cisplatin
• Double-edged sword: promoting DNA damage
(which you want) also creates mutations
hence providing escape routes
to develop resistance
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Resistance to apoptosis
• Disruption of apoptotic pathways is a hallmark of
cancer and a major obstacle to chemotherapy
• Bcl2 family of genes (homeostatic balance of proand anti-apoptotic activities)
• Many (all) anticancer drugs affect alternative splicing
of apoptotic genes (not always going towards proapoptotic variants) Shkreta et al. 2008. Mol Cancer Ther 2008;7(6):1398–409
• DNA damage-mediated shifts in Bcl-x splicing
through modification of splicing regulators
Shkreta et al. 2010 JBC in press.
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microenvironment
Solid tumor
Bad vascularization
Hypoxia
Glycolysis
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Acidification
Hypoxia
• Hypoxia reduces efficiency of alkylating
agents and platinium-based
compounds, bleomycine
• Overall, hypoxia causes resistance to
anticancer therapies and leads to
selection of even more resistant
phenotypes.
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Hypoxia - HIF
• HIF-1 and HIF-3 modulate transcription of gene
clusters involved in adaptation and survival.
• HIF-1 overexpressed in cancer and good marker for
resistance and inefficient anticancer treatments
• HIF-1 expression is
a drug target
• HIF-1 and HIF-3 are
alternatively spliced
and variants displays
distinct activation
properties
(on VEGF, for example)
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Hypoxia - VEGF
• VEGF is required for revascularization of
tumor environment
• Targeting VGEF
with Bevacizumab
prevents binding
to its receptor
VGEFR
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PROTEIN AND mRNA SPLICING PRODUCTS OF VEGF-A
GENE STRUCTURE
EXON
1
2
3
4
5
6
7
8
SPLICING VARIANTS
PRO-ANGIOGENIC
ANTI-ANGIOGENIC
VEGF-A206
VEGF-A189b
VEGF-A189
VEGF-A183
VEGF-A165
VEGF-A183b
VEGF-A165b
VEGF-A148
VEGF-A145
VEGF-A121
VEGF-A145b
VEGF-A121b
PROTEIN STRUCTURE
EPITOPE
recognized by
BEVACIZUMAB (AVASTIN)
(in exon 4)
(modified from Harper and Bates 2008 Nature Review Cancer)
Modulator of
ANGIOGENIC or
ANTI-ANGIOGENIC
function
Glycolysis
• Hypoxic cells switch to glucose
metabolism through glycolysis
• PKM1 elicits oxydative phosphorylation
• PKM2 splice variant is induced by
hypoxia and elicits aerobic glycolysis
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PKM splicing regulation
exon 9 is skipped
From David et al. 2010 Nature 463, 364-368
Q-CROC - Sherbrooke
• Clinical samples
• Splice isoform profiling for all the genes mentioned in
this meeting and more
Sherbrooke - RNA platform
For info; roscoe.klinck@usherbrooke.ca
A membrane story - HER2
Herceptin does not work for 30-50%
of breast cancer patients
The majority of cancers
responding to herceptin become
resistant within 1 year
∆Her2 is a splice variant
that has increased transformation
potential. Cells that express ∆Her2
are less sensitive to herceptin
(Mol Carcinog. 1998 23:62-8)
Her2∆15 can be programmed with
antisense oligos and it works as a
dominant negative
(Wan et al. Intl. J. Cancer 2009 124:772-7)
Acknowledgements
Luli Shkreta
Mechanisms
• Genetic polymorphisms in many of these
genes are associated with intrinsic and
acquired resistance
• What are the chances that this involves
differences in splicing control ?
+
• Anticancer drugs promote genomic instability
• Genomic instability means mutations
• Mutations often mean changes in alternative
splicing
Proteinase-cell adhesionsignalling
• ADAM-17 and EGFR pathway
• Inhibiting ADAM expression sensitizes
prostate cancer cells to radiation and
chemotherapy
• TRAIL and 5-FU enhance ADAM-17mediated shedding of EGFR and activation of
pro-survival pathways (resistance to TRAIL)
• Secreted and membrane-bound splice forms
for each of the 25 ADAM genes
Integrins
• Integrin a4b1 mediates adhesion to
extracellular matrix and stromal cells
leading to cell-adhesion mediated drug
resistance (selection for drug resistance
upregulates a4b10
• Lots of alternative splicing for integrins
which changes ligand binding,
localization, signalling
CDK10
Endocrine resistance in breast
cancer
• Tamoxifen to inhibit growth of ER-positive
breast cancer
• Intrinsic resistance = lack of ER, inactive
alleles of cytochrome P450 2D6
• Acquired resistance = anything along the ER
pathway (change in expression of cyclins,
myC, CDK, RB , Bcl-2 family members) next
slide
Molecular mechanism of
endocrine resistance
• Figure 3 in musgrove nat review cancer 2009
• Although based on in vitro studies, change in
level, structure (splicing) could in principle
trigger resistance. Confirmed to some extent
by complex expression signatures from
patient biopsies.
• These changes are stimulated by co-treating
with compounds that promote genetic
instability (mutations).
Endocrine resistance in breast
cancer
• HER/neu overexpression targeted by
antibodies (herceptin)
• CDK10 drops in expression associated
with resistance of breast cancer to
tamoxifen
• (Iorns et al Cancer Cell 2008)
acidification
• Increased glycolysis produces lactic acid, pH drops, H+
transported in microenvironment, breakdown cellular matrix
• Low pH reduces cellular uptake of weakly basic drugs (e.g.,
vinblastine, vincristine, doxyrubicine). Therapeutically changing
pH improves efficiency of of the above drugs and paclitaxel,
topotecan.
• Low pH changes expression of splicing regulators and switches
splicing in favor of a more angiogenic form of VEGF
• HIF1 induces CA IX (cancer-isoform). CA IX reduces pH further.
This isoform is associated with resistance to radiotherapy and
treatment in many cancers.
• AS-CA IX (found in normal tissues but less or not in cancer)
antagonizes activity of CA IX.
• Why not change splicing in favor of AS-CA IX ?