Drosophila: Modelling Metastasis
Misexpression and Overexpression
Requires new transgenic strain for each possible
combination of tissue-specific enhancer and
gene of interest
Some combinations are lethal/sterile so stable transgenic stocks cannot be
maintained
Binary Misexpression System: GAL4/UAS
GAL4 activates transcription of any
coding sequence downstream of its
Upstream Activating Sequence (UAS) – A
single UAS line can be crossed to various
GAL4 lines to examine consequences of
tissue-specific expression
UAS is a neutral promoter – idling, doesn’t haven enhancers but when GAL4 binds to
it, it activates
Tissue-specific enhancer GAL4 transgenic flies crossed to UAS-coding sequence
transgenic flies
Coding sequence in tissue-specific pattern in doubly transgenic offspring
Ternary Misexpression System
GAL80 binds to GAL4 preventing it from
binding to the UAS – no expression from
the UAS
Stage-specific/inducible enhancer GAL80
– tissue specific enhancer GAL-4
transgenic flies crossed to UAS-coding sequence transgenic flies
Coding sequence expressed in stage and tissue specific pattern in triply transgenic
offspring
Metastasis
1) Cooperation – between oncogenes and tumour suppressors (multi-hit process)
2) Invasiveness – tumour cells acquire ability to grow through extracellular matrix and
basement membranes
3) Microenvironment – involves interactions between tumour cells and environment
(some failure of the tumour cells to appropriately interact with the environment)
System: Modelling Metastasis (Pagliarini and Xu, 2003)
, Formation of benign tumours and if there is an
extra mutation – tumours begin to move around
and grow beyond territory it was restricted to
Ternary misexpression + site-specific
recombination (to induce mitotic recombination)
- RASV12 = oncogene, constitutively active, gives
tumours/overgrowth, controlled by UAS –
only activated when GAL4
interacts, inhibited by GAL80
(+GFP)
- (Actin) Act-GAL4 = ubiquitous
GAL4 – expressed in all cells
- (Tubulin) Tub-GAL8- = ubiquitous
GAL80 – expressed in all cells
- Net effect of all these
transgene should be no tumours
- Scrib is a tumour suppressor
(mutant on red chromosome, wild-type of blue chromosome – effects when
homozygous)
- eyFLP = eye-disc specific FLP
- FLP activates – recombination – producing 2 daughter cells after mitosis
One daughter cell has wild-type phenotype
One daughter has all the bad stuff – Does not have Tub-GAL80, homozygous for
Act-Gal4, Ras activated
With single Ras or scrib/scrib mutant cells (no Ras/scrib in cell) – overgrowth
restricted to the eye, visual cortex in brain – localisation of system
Double Ras or scrib/scrib mutant cells – invasive outgrowth
Ras and scrib/scrib mutants cooperate to induce metastatic overgrowth
Modifier screen for gene cooperation (Wu et
al., 2010)
Used the above system to screen for
equivalents of scrib – those that can modify
Ras activity
Flies with backround (basic one), mutagenize
and cross, observe phenotypes
If chromosomes are not on the same
chromosome – dominant enhancers as
suppressors
, New mutations on same chromosome as FRT – same effect as scrib – induces
metastasis
Deep orange (promotes endosomal trafficking) = metastatic enhancer of Ras
- Pigment synthesised in abdomen and carried to eye – so eye colour also involves
cellular transport
- Loss of Endocytotic activity promotes metastasis
Gene cooperation in drosophila (Herranz et al., 2012)
Wing disc system, EGFR induces benign overgrowth – under
control of UAS
Ternary system – GAL80 is temperature
sensitive, 18 activates GAL40, inhibits EGFR, at 29, GAL80 inhibited, UAS drives
EGFR
- Apterous restricted to dorsal wing
disc specific driver(GFP green)
Not screen – tested for a number of candidate cooperating factors
miRNA Bantam – implicated in control of cell proliferation
Bantam/EGFR co-expression induces metastatic growth
Bantam not conserved in mammals – but bantam targets SCOS36E, negative
regulator of JAK/STAT signalling
Human homologue of SCOS36E also shown to act as tumour suppressor when EGFR
signalling elevated
Invasiveness
Tumour cells must penetrate the
basement membrane/ECM
Normal cells restricted to tissue
of origin by ECM and basement
membranes
Cancer cells form cell protrusions
(inadopodia) by localised assembly of actin cytoskeleton
Invadopodia invades physically by penetrating the membrane and chemically
secreting matrix metalloproteases (and other proteases) which digest the basement
membrane facilliatiting metastasis and the crossing of multiple tissue layers
Drosophila model of metastasis
Misexpression and Overexpression
Requires new transgenic strain for each possible
combination of tissue-specific enhancer and
gene of interest
Some combinations are lethal/sterile so stable transgenic stocks cannot be
maintained
Binary Misexpression System: GAL4/UAS
GAL4 activates transcription of any
coding sequence downstream of its
Upstream Activating Sequence (UAS) – A
single UAS line can be crossed to various
GAL4 lines to examine consequences of
tissue-specific expression
UAS is a neutral promoter – idling, doesn’t haven enhancers but when GAL4 binds to
it, it activates
Tissue-specific enhancer GAL4 transgenic flies crossed to UAS-coding sequence
transgenic flies
Coding sequence in tissue-specific pattern in doubly transgenic offspring
Ternary Misexpression System
GAL80 binds to GAL4 preventing it from
binding to the UAS – no expression from
the UAS
Stage-specific/inducible enhancer GAL80
– tissue specific enhancer GAL-4
transgenic flies crossed to UAS-coding sequence transgenic flies
Coding sequence expressed in stage and tissue specific pattern in triply transgenic
offspring
Metastasis
1) Cooperation – between oncogenes and tumour suppressors (multi-hit process)
2) Invasiveness – tumour cells acquire ability to grow through extracellular matrix and
basement membranes
3) Microenvironment – involves interactions between tumour cells and environment
(some failure of the tumour cells to appropriately interact with the environment)
System: Modelling Metastasis (Pagliarini and Xu, 2003)
, Formation of benign tumours and if there is an
extra mutation – tumours begin to move around
and grow beyond territory it was restricted to
Ternary misexpression + site-specific
recombination (to induce mitotic recombination)
- RASV12 = oncogene, constitutively active, gives
tumours/overgrowth, controlled by UAS –
only activated when GAL4
interacts, inhibited by GAL80
(+GFP)
- (Actin) Act-GAL4 = ubiquitous
GAL4 – expressed in all cells
- (Tubulin) Tub-GAL8- = ubiquitous
GAL80 – expressed in all cells
- Net effect of all these
transgene should be no tumours
- Scrib is a tumour suppressor
(mutant on red chromosome, wild-type of blue chromosome – effects when
homozygous)
- eyFLP = eye-disc specific FLP
- FLP activates – recombination – producing 2 daughter cells after mitosis
One daughter cell has wild-type phenotype
One daughter has all the bad stuff – Does not have Tub-GAL80, homozygous for
Act-Gal4, Ras activated
With single Ras or scrib/scrib mutant cells (no Ras/scrib in cell) – overgrowth
restricted to the eye, visual cortex in brain – localisation of system
Double Ras or scrib/scrib mutant cells – invasive outgrowth
Ras and scrib/scrib mutants cooperate to induce metastatic overgrowth
Modifier screen for gene cooperation (Wu et
al., 2010)
Used the above system to screen for
equivalents of scrib – those that can modify
Ras activity
Flies with backround (basic one), mutagenize
and cross, observe phenotypes
If chromosomes are not on the same
chromosome – dominant enhancers as
suppressors
, New mutations on same chromosome as FRT – same effect as scrib – induces
metastasis
Deep orange (promotes endosomal trafficking) = metastatic enhancer of Ras
- Pigment synthesised in abdomen and carried to eye – so eye colour also involves
cellular transport
- Loss of Endocytotic activity promotes metastasis
Gene cooperation in drosophila (Herranz et al., 2012)
Wing disc system, EGFR induces benign overgrowth – under
control of UAS
Ternary system – GAL80 is temperature
sensitive, 18 activates GAL40, inhibits EGFR, at 29, GAL80 inhibited, UAS drives
EGFR
- Apterous restricted to dorsal wing
disc specific driver(GFP green)
Not screen – tested for a number of candidate cooperating factors
miRNA Bantam – implicated in control of cell proliferation
Bantam/EGFR co-expression induces metastatic growth
Bantam not conserved in mammals – but bantam targets SCOS36E, negative
regulator of JAK/STAT signalling
Human homologue of SCOS36E also shown to act as tumour suppressor when EGFR
signalling elevated
Invasiveness
Tumour cells must penetrate the
basement membrane/ECM
Normal cells restricted to tissue
of origin by ECM and basement
membranes
Cancer cells form cell protrusions
(inadopodia) by localised assembly of actin cytoskeleton
Invadopodia invades physically by penetrating the membrane and chemically
secreting matrix metalloproteases (and other proteases) which digest the basement
membrane facilliatiting metastasis and the crossing of multiple tissue layers
Drosophila model of metastasis
