AACR Annual meeting 2013
Abbreviations:
ECM: Extracellular Matrix
EMT: Epithelio-Mesenchymal transition
TME: Tumor Microenvironment
7/4, 7am, MEET-THE-EXPERT SESSION
Jeffrey Wrana
Dysregulated morphogenesis and polarity in cancer
Polarity is a key control of morphogenesis, which is controlled by a limited number of pathways
(TGFb, WNT, Notch…). Dysmorphogenesis occurs during cancer development, but little is known
about the integration of morphogen pathways into higher order networks that define the contextual
cues driving cancer progression. The tumor stroma plays a critical role in cancer progression, but how
it promotes metastasis is poorly understood.
Fibroblast conditioned media (ACM)
MDAK cells
activated breast cancer cells (BCC)!
Fibroblasts secrete exosomes sufficient to promote BCC protrusive activity, motility and metastasis.
Smurf1 & 2 (ubiquitine ligases) are regulators of cell polarity, protrusive activity and BCC motility.
Their KO by siRNA inhibits ACM activity. As they are PCP (planar cell polarity) regulator, they verified
if the ACM-induced motility was mediated through the core PCP pathways. Downregulation of the
core PCP component reduces metastasis, suggesting their specific role in metastasis.
Wnt ligands are key regulators of the PCP pathways. Their KO in fibroblast has surprisingly no effect.
On the contrary, their KO in BBC inhibits ACM activity! It indicates that ACM stimulates autocrine
Wnt pathways that activate PCP signaling during BCC protrusive activity and motility.
To identify the factors that promote autocrine Wnt-PCP signaling in BCCs, they separated the purified
ACM in single activity fractions. Among several exosomes markers, the loss of Cd81-positive
exosomes inhibits ACM-induced BCC protrusive activity and motility. Cd81 KO tumors do not present
metastasis, but grow as fast as a Cd81+ tumor. Indeed, CD81 expression is upregulated in human
breast tumor-associated stroma.
 Exosome-driven metastasis is dependent on the core PCP pathways. Cd81-positive exosomes
are internalized by BBC with Wnt11. It reveals a key role for fibroblasts-derived exosomes in
mobilizing autocrine Wnt-PCP signaling in BCC to stimulate invasive behavior and metastasis.
Luga, Wrana et al, Cell 2012
7/4, 3pm, MINISYMPOSIUM
TISSUE ORGANIZATION, MECHANICAL
MICROENVIRONMENT
FORCES,
AND
3-D
MODELING
OF
THE
Thimothy Fessenden
Dynamic multicellular mechanics controlling protrusion in 3D
Tension maintained across the actin cortex mechanically restrict protrusions, and may be transmitted
across multiple cells via adherens junctions, as it exists in epithelia. How is tissue organization
disrupted to allow invasive behavior?
He studied the MDCK acini morphogenesis upon HGF exposure as a model for rupture of epithelial
integrity and protrusion.
Jonathan Celli
Microrheology studies in 3D pancreatic tumor models
Pancreatic tumors are very rigid and have hyperdense stroma, which increase invasiveness and
reduce drug delivery. Key questions about the role of mechanical microenvironment remain
unanswered. Cocultures (fibroblasts and cancer cells) are embedded in collagen matrix containing
fluorescent tracer probes. The Brownian motion of the probes is used as a reporter of spatiotemporal changes in viscoelastic properties of the ECM and correlated with the architecture changes
and the cytotoxic response.
Preliminary results show heterogeneity in ECM rigidity and a role from fibroblasts to restore ECM
uniformity.
Hal Crosswell
The 3DKUBE segregated co-culture system
Coculture of MCF7 spheroids in IrECM next to hFFB fibroblasts in scaffold to mimic the stroma, and
both perfused with controlled flow rates and cycling times. This system promotes spheroid
maintenance and growth, and is applicable across multiple different tumor types. The assay with
PI3K inhibitors show a reduced activity in the 3D perfusion model compared to the 3D and 2D
models.
8/4, 6:45am, MEET-THE-EXPERT SESSION
Andrew Futreal
Inter- and intratumor heterogeneity in human cancer
In our sequencing generation, it is nowadays possible to extract either a single mutation or the whole
genomic of every unique cancer. However, the multiple heterogeneity in cancers increases the
complexity of deducing information from cancer genome sequencing.
1) Heterogeneity between the same cancer types
For example chondrosarcoma can be mutated on different genes: IDH1/2, TP53, Hedgehog pathway,
Col2A1, etc. And even for one mutated gene, it often exists several possible mutations: Col2A1 is
often truncated, but also presents missense mutation but never silent mutation.
2) Heterogeneity within the same tumor
A multiregion exome sequencing in CCRCC revealed that 65% of mutations from a single biopsy are
heterogeneous. The mutations can be ordered and draw a tree of mutations evolution. Moreover,
mutations can have different impacts on cell phenotype.
Gerlinger et al, 2012, NEJM
3) Heterogeneity over time
Exome sequencing of longitudinal sample indicate the % of mutated reads of each mutation (Variant
Allele Frequency, VAC) and can reveal a shift in subclone distribution during a tumor treatment.
There are 4 possible evolutions during a treatment:
- No evolution
- Appearance of a dominating subclone
- Developing of a new subclone which was absent at the early stage
- Completely different evolutions between the early and late stages.
 Is the complexity subclonal architecture a prognostic? Where do the drivers and targets live?
Does it matter? Could these types of analyses be reduced to clinical practice? Are there
patterns of timing metastasis?
8/4, 1pm, MAJOR SYMPOSIUM
THE RUTH SAGER MEMORIAL SYMPOSIUM: CANCER HETEROGENEITY
Elaine Mardis
The impact of therapy on tumor heterogeneity
Elaine Mardis bring complementary information to the Andrew Futreal presentation.
1) They hypothesized that an older mutation is more frequent, and consequently the study of
mutation frequencies in primary tumor and metastases could let draw the tree of metastatic
evolution of the cancer. They show the result of a case study from patients near to death
with multiple metastases.
2) They compared the VAC of breast tumor treated with different drugs to define the effect of
drugs on cellular heterogeneity.
3) They compared the VAC of xenografts from primary tumor and metastasis of the same tumor
to analyze the preservation or alteration during metastasis and xenografts process. Their
work reveal a good correlation between the changed observed from the primary tumor and
metastasis, and the changed observed between the corresponding xenografts. This could be
a good mean to extend the comparison of primary tumor and metastases to better evaluate
the resistance of the tumor.
Peter Campbell
Patterns of subclonal heterogeneity across tumor types
They studied the impact of driver mutation on the patient prognosis, in a cohort of patients with
myelodysplasic tumors.
First, they analyzed the frequencies of driver mutation in the tumors. They found 3 classes of driver
mutation: known oncogenic mutations, possible oncogenic mutation, and variants with unknown
significance. Around 40% of the patients have more than one driver mutation.
Second, they analyzed the association of mutated genes. It appeared that some mutated genes are
strongly associated, but some other mutations never happen together.
Thirdly, they verified if the driver mutation accumulation could decrease the prognostic. Indeed, the
more patients accumulate driver mutations, the more the risk of bad prognostic is soon. However,
there is no difference in survival between a clonal and a subclonal mutation of the same driver
mutation.
Personal remarks
All those talks on cancer heterogeneity were very enriching and remind the complexity of the
tumors. It is likely that the cellular heterogeneity mimicked in MCTS is largely reduced compared to
the one found in tumors.
If we consider that two genetically different cells might have two different phenotypes, two tumors
with different heterogeneity might evaluate in different ways. From my point of view, those studies
are sometimes far from the cellular process and the biological steps of tumor development and
evolution.
8/4, 3pm, MINISYMPOSIUM
MICROENVIRONMENT DRIVING TUMOR INITIATION
Tracy Pritchett
Novel conditional knockout of VHL in the kidney leads to precancerous lesions that exhibit an
inflammatory response
As the VHL gene is mutated in 80% of renal cancers, they produced transgenic mice with a
conditional knockout of VHL in the kidney. They used the CreLoxP method, with Cre expressed in
kidney collective ducts. Mice presented precancerous lesions with infiltration, clear cells, epithelial
cells pill up, high proliferation, high vascularization and high fibrosis.
One major function of VHL is to act as en E3 ubiquitin ligase. Consequently of the KO, its major target
HIF is high, as are HIF targets Glut1 and CAIX. They contribute in fibrotic defects. Pritchett and
coworkers produced double KO of VHL and HIF, and they observed a great reduction in lesions
appearance.
This model reproduces renal precancerous and cancerous lesions, and thus can be used to better
understand the mechanisms of switch from fibrosis to cancer.
Aubie Shaw
Myeloid cells promote fibroblast invasion and lead mammary adenocarcinoma cell invasion
Alenxander and Friedl 2012
Fibroblasts and macrophages are important for extravasation processus. Macrophages regulate
invasion and metastasis. They are localized near vessels. CD4+ cells promote macrophages that
participate to tumor progression. It appears essential to inhibit macrophages to limit metastasis. On
the other hand, fibroblasts lead collective cell migration through ECM remodeling (Khalil and Friedl
2010, Gritsenko, Ilina and Friedl 2012). Shaw and coworkers wanted to verify the hypothesis of
collaboration between fibroblasts and macrophages. They separated myeloid cells, carcinoma cells
and fibroblasts from primary cells from mice and cocultivated them in collagen or IBME matrix. They
observed fibroblasts migration towards specific myeloid cell types. It appeared that many myeloid
cells secreted factors to promote fibroblasts invasion. FGFR1K is a crucial factor for migration.
Indeed, its inhibition blocks fibroblasts migration. They wondered if there was a direct cell-to-cell
interaction. Ongoing studies are identifying specific subpopultions of tumor-associated myeloid cells
participating to collective cell migration.
Valentino Audrito
Extracellular form of NAMPT (eNAMPT) contributes to creating a proinflammatory environment in
chronic lympocytic leukemia
NAD is a signaling molecule regulating metabolic enzymes such as NAMPT. It acts as a cytokine. Its
secretion is increased in leukemia, making this biosynthetic pathway an attractive target for cancer
treatment. The aim of their work is to test whether eNAMPT played a role in shaping the leukemic
environment, by generating proinflammatory conditions.
NAMPT is more secreted after B lymphocytes activation. However, NAMPT does not act directly on
fibroblasts. It enhances differentiation and phagocyte activity of nurse-like cells. The data suggest
that eNAMPT contributes to the formation of a proinflammatory environment.
Adriana Albini
Innate immunity driving tumor angiogenesis: the role of natural killer cells (NK) in NSCLC
NK represent 5-10% of lymphocytes. They use granzime and perforin killing means. But among their
different subsets, one of them, CD56+/++ and CD16- does not kill: it produces cytokines and
participates to tissue construction. CD56+ NK predominate in cancers, while they are low in blood
and adjacent tissues. The analyses of biopsies and blood from patients revealed the production of IL8
and INFγ by NK. Those cytokines stimulate endothelial cells chemotaxis and formation of capillarylike structures in vitro. It also appeared that TFGb , associated with a poor prognostic, mediated NK
recruitment towards an angiogenic activity.
To conclude, CD56+ NK could be targeted to prevent angiogenesis. Could NK be a marker of
prognosis and complication?
David Nguyen
Biologically distinct mammary tumors develop from Trp53 null tissue transplanted to juvenile versus
adult gland
The risk of breast cancer is higher during puberty, due to stem cells expansion. They investigated the
impact of the age of transplantation on tumor development. They transplanted Trp53 null mammary
fragments into inguinal fat pads of 5 week-old or 10 week-old mice. Pubertal mice presented bigger
tumors with higher mitotic indexes. 80% of their tumors were ER+. A microarray analysis revealed a
luminal transcriptional program. To conclude, factors determining breast cancer molecular subtype
may not be epithelial autonomous, but can be influenced by the host environment.
Tomohiro Umezo
Hypoxia modulates tumor exosomes, which function as mediators of angiogenesis
Exosomes are mediators of communication. They contain proteins, mRNA, miRNA, etc. The aim of
the study was to clarify whether hypoxia promote angiogenic activity through tumor exosomes.
To answer the question, they cultivated endothelial cells with conditioned media from normoxia
(20% O2) or hypoxia (1% O2) cancer cells, then analyzed exosomes by using nanoparticules tracking
and exosome miRNA profiling. Hypoxic exosomes promote tube formation of endothelial cells.
miR210 was elevated in hypoxic conditions. It regulates the target genes of a hypoxic response in
endothelial cells. Hypoxic exosomes play a role in inducing angiogenesis.
9/4, 7am, MEET-THE-EXPERT SESSION
Chi Van Dang
Links between Metabolism and cancer
Cells are replicating bioreactors. In homeostasis condition, they use amino acids, glucose and fatty
acids by oxydation to produce ATP and waste. In growth condition, growth factors induce glycolysis
and Krebs cycling to increase cell biomass and lead to cell division. Glucose (Glc), but also glutamine
(Glt) can feed the Krebs cycle to produce lipids, nucleic acids and ATP. Growth factors are necessary
to activate cell growth. They activate the gene MYC, which can activate the three m, r and tRNA and
numerous metabolic pathways. P53 and Art keep MYC quiet enough to control proliferation except if
they are mutated.
1) Can metabolism cause cancer? It has been shown that Glc deprivation induced mutant Ras.
Indeed, Ras or Braf mutation can activate the Glc1 transporter. Many other metabolic genes
are known to be altered in cancers. (Shih, Levine, NRC, 2013) To conclude, oncogenes and
tumor suppressor genes are more and more linked with metabolic genes, increasing the
complexity of the genes network.
2) Does it exist fragility in cancer cells? Nutrients regulate ribosome biogenesis:
Glc -> RAS –I Dot6 –I Ribigene expression
Glt -> TORC1 –I Ted6 –I Ribigene expression
Dot6 and Ted6 are often mutated in cancer, because cancer cells are addicted to Glc and
Glutamine but still need Ribigene expression. Myc enhance Glt oxidation x4, while Glc
oxidation is only increased x1.2. Is the Glt pathway a possible target? GLS1 converts Glt and
Glutamate. GLS+/+ mice develop bigger tumors than GLS-/- mice. They treated mice with
BPTES, a GLS inhibitor, and reduced the tumor development resulting in an extended life of
animals.
Dang gave another example with the pyruvate/lactate pathway. Le et al, PNAS 2010.
3) Is there a therapeutic window? Mutations induce new activities specific to cancer cells, and
therefore, that can be targeted. For example, liver cells are GLS2 + while liver cancer cells
have GLS2 low expression and GLS1 very high expression.
4) Are there oncogenic alterations of the circadian molecular clock? Organs metabolisms are
synchronized with food arrival, activities, light, etc. Does Myc disrupt the cycling expression
of metabolic genes? Indeed, under Myc stimulation, expression of metabolic genes becomes
permanent and high. This difference in levels of expression opens a new therapeutic window.
9/4, 8am, MAJOR SESSION
MITOTIC CHECKPOINTS DRIVING CANCER THERAPEUTIC STRATEGIES
Timothy Mitchison
Is mitosis the target of “antimitotic” cancer drugs?
In tumor, only 2-5% of the cells are in S phase, while there are 40% in bone marrow! 2D cultures are
bad predictors of patient response for cytotoxic drugs. Nowadays, two types of drugs are used:
- Anti-microtubule drugs which also affect interphase cells
- Anti-mitotic drugs which only affect proliferative cells
Anti-microtubule drugs induce SAC activation and block cell in anaphase or in G1, leading to cell
death or senescence. Consequently, the mitotic arrest becomes toxic only if a normal mitotic exit
fails.
They used HT1080 expressing Fucci reporter to visualize the behavior and death of injected cells in
mice. They revealed a concentration-dependent effect of taxol treatment.
Low concentration
Aneuploidy with mitotic arrest
mitotic arrest
High concentration
interphase bundle
The interphase bundle could be a therapeutically relevant mechanism. However, taxol
pharmacokinetics are complicated to control and it is only its accumulation that could raise an
effective action.
They also showed that anti-mitotic drugs have anti-vascular effects : they disrupt established tumor
vasculature and inhibit new blood vessel formation.
Peter Senter
Tumor targeting with anti-mitotic monoclonal antibody drug conjugates
Senter presented various antibodies targeting tumor cells and used as mean to deliver drugs close to
tumor. They precised their results obtained with the MMAE linked to an antibody.
Guido Kroemer
Targeting tetraploidy for cancer therapy by pharmacological and immunological strategies
They made a drug screening on cocultures of diploid cells and tetraploid H2B-GFP cells. They
analyzed the proliferative effect and the evolution of the diploid/tetraploid rate.
The conclusion of their work revealed that tetraploid cells are immunogenic in vivo, and taxanes
induce tetraploidy in human cancers. As their elimination correlate with chemotherapeutic effects,
they might be used as predictive biomarkers.
9/4, 3pm, MINISYMPOSIUM
NEW PATHWAYS IN TUMOR METASTASIS
Rosandra Kaplan
Breaking metastatic dormancy during surgical resection
After surgical resection, bone-marrow derived cells are activated and mobilized at the resection site.
There, they induce a local inflamed TME and provide survival signal to the seeding tumor cells.
Indeed, vasculogenic monocytes, hematopoietic progenitor cells and endothelial progenitor cells are
elevated in blood of mice which underwent tumor resection, while there are not in control mice
which underwent a surgery without resection. Those concentrations of circulating cells are
correlated with bad prognostic.
They observed that surgical resection of primary tumor in mice enhanced metastasis! It is partly due
to an increase in mobilized cells. They showed that targeting the circulating cells with Pazopanib
prevented the surge in bone-marrow-derived cells into the circulation and abolished the enhanced
metatatic spread in mice undergoing surgical resection. It provided a significant prolongation of life.
Consequently, inhibiting the mobilization of bone marrow-derived cells following surgery could
reduce the metastatic capacity of disseminated tumors.
9/4, 5pm, FORUM
THE ROLE OF STROMA IN PANCREATIC CANCER
Ben Stanger
In pancreatic cancer, cancer cells are minor components of the tumor. As EMT and dissemination
precede pancreatic tumor, most tumors have already spread at the time of the detection, resulting in
high mortality.
The TME is composed of many cell types, it is necessary to understand their behavior in order to
target them. The team established a mice model allowing the lineage of KRAS mutated cells. It
appeared that stromal cells are derived from KRAS epithelial cells. They present different stage of
EMT: regular compartment of E-Cadherin expressing epithelial cells, mesenchymal cells expressing ECadherin, or fully stromal cells.
Do the stromal cells have a positive or negative influence on cancer cells?
The Hh pathway is strongly activated in pancreatic cancer cells. Hh inhibition increases vessels
density and drug delivery (pancreatic cancer is avascular). To answer the question, they produced
shh KO mice with KRAS mutation. Tumors stayed undifferentiated with reduced fibroblastic stroma.
They were higher vascularized and the drug had a better penetrance. Surprisingly, tumors are more
dangerous because the cancer cells proliferated more rapidly and it resulted in faster death! The
good point is that the tumors become more sensitive to anti-angiogenic drugs.
 The better understanding of tumor microenvironment is necessary to understand the
perturbation of the expected consequences of a treatment.
Philip Beachy
With a lineage of basal cells of the bladder, Beachy and coworkers showed that bladder tumors arise
from a single urothelial basal cell.
10/4, 8am, PLENARY SESSION
CANCER EVOLUTION AND RESISTANCE
Jeff Engelman
Resistance is due to:
- Gene amplification/overexpression
- Bypassed pathways
- Defect growth/apoptosis/EMT/etc
The two first phenomena are the more potent to be inhibited by drugs combination.
Engelman and coworkers made a drug combination screen on EGFR inhibitor resistant cells. The
EGFR inhibitor resistant cells are produced by multiple passaging in increased drugs concentration.
78 drugs are tested, and some combination appeared to reduce the resistant cell viability. Their
validation was made on patient derived models (cell lines and xenograft).
From this study, it appears that the tumor cells heterogeneity majors the challenge to overcome
resistance. Indeed, the cells cultured from biopsy develop depending on the drug, and it can make a
new resistant cell population rises from the sensitive cell background.
 If the sensitive cells are not killed in totality, they grow up again during treatment release,
making the patient sensitive to the drug again. But while, resistant cells grew and the
response is less efficient. This might explain why remissions only last few months.
 A protocol would be to alternate between different combinations without any interruption in
the EGFR inhibitor treatment.
Todd Golub
Microenvironmental contributions to cancer drug resistance
Does tumor microenvironment mediate tumor resistance?
They studied the effect of fibroblast on cancer cell resistance to several drugs, by cocultures with
cancer cells and fibroblasts. It appeared that some fibroblast rescued melanoma treated with
Vemurafenib. This rescue was reproduced by adding the fibroblast-conditioned medium to the
cancer cells.
 This underlines the great importance of the secretome!
It has been shown that HGF secretion is sufficient to induce resistance. If stromal cells secrete HGF,
the downstream pathway is activated in cancer cells, giving them resistance! HGF inhibitors and
Verumafenib combination should be more efficient to avoid tumor resistance. It will be tested on
patients soon.
10/4, 11am
TARGETTING THE CELL CYCLE
Marco Malumbres
Targeting mitosis for cancer therapy
As CDK4/6 inhibitors arrest tumor cells in G0 or G1 and do not induce cell death, mitosis should be a
better target to induce cell death. Microtubules poisons might be good candidates but mitotic index
is often low in tumors, and cells escape by mitotic slippage. It appears essential to inhibit the mitotic
exit to make mitotic poisons efficient. Malumbres and his team are trying to indentify some drugs
with this effect.
Nat Rev Clin Oncol, T. Fojo 2011
Mitchison 2009
Malumbres 2010
Mariano Barbacid
Targeting KRAS cancers: CDKs andf other mechanisms
Growth factors activate the RAS pathways, implicated in many cell activities. KRAS mutations turn the
RAS pathway ON. KRAS is not druggable but its downstream effectors are. Two questions are not yet
answered:
-Which kinases are essential?
-Which kinases are selective for targeting KRAS?
1) They targeting different kinases by conditional KO in KRAS-mutant mice models, and analyzed the
absence, the partial or the complete response of the target inhibition against the KRAS lung
adenocarcinoma.
Target
Tumor development
Toxicity
A-Raf
Yes
B-Raf
Yes
C-Raf
NO!
NO!
CDK2
Yes
CDK4
NO!
Diabetes
CDK6
Yes
 C-Raf and CDK4 are essential for the KRAS to initiate the tumor.
2) With a second model of conditional KO of target, they induced the tumor and then analyzed the
remission by the inhibition of the target.
With CDK4/6 inhibitor, the tumor does not disappear even if its volume decreases.
With C-Raf inhibition, the tumor remains as big but become PET negative (dormant tumor) and the
survivance is reestablished. They hypothesis this partial result was due to an inhibition and not a
absence of the proteins. As those proteins are necessary for development, they produced deadkinase that are totally inactive. The first mice with dead kinase seem healthy, meaning that the
kinases have non-kinase activities necessary for the embryonic development. Further studies on their
role in tumor development are ongoing.