Peyton Rous

1
Peyton Rous
Reported his namesake Rous Sarcoma Virus In 1911
Nobel Prize 1966
2
SRC READING MATERIAL
CLASSIC REVIEW Thomas and Brugge. Ann Rev.
Cell Dev. Biol 13 513-609, 1997 EXCELLENT
OVERVIEW OF PRINCIPAL SRC FUNCTIONS

Frame, M.C. J. Cell Sci. 113 989-98, 2004 a
reasonable update
ONCOGENE, volume 23 Oct. 18, 2004-The whole
issue is devoted to Src family Structure function
Src and Cancer Biology Summy, JM and Gallick
GE. Cancer Met Rev. 22 337-358, 2003 Src and
the Clinic Summy JM and Gallick, GE Clin Can
Res 12 1398-401, 2006 Src inhibitors IN the
clinic Kopetz et al., Clin. Ca Res 13 7232-6,
2007 Src and EMT Avizienyte E, Brunton VG,
Fincham VJ, Frame MC. The SRC-induced
mesenchymal state in late-stage colon cancer
cells. Cells Tissues Organs. 17973-80, 2005.
3
SRC READING MATERIAL

Src and Survival and Angiogenesis -Schlessinger
J. Cell. 100(3)293-6, 2000. Src, inflammation
and metastasis-Park et al., Cell Tissue Research
2009, 335 249-59
4
Non Receptor Tyrosine Kinases
SH4
SH3
SH2
SH1
Src, Fyn, Yes, Blk, Fgr, Hck, Lck, Lyn,
Frk/Rak Iyk/Bsk
Y
unique
catalytic
Csk
PH
TH
Tek
Syk/ZAP
Focal adhesion targeting
Integrin binding
FAK
Paxillin binding
Talin binding
5
Expression of Src family members

• Src, Yes, Fyn, Yrk-ubiquitous
• Fyn has an alternate splice form found in T cells
  only
• Yrk is expressed only in chickens
• Lyn-Brain, B cells, myeloid cells-recently
  prostate
• Two alternately spliced forms
• Hck-myeloid cells (two translational start sites)
• May contribute to CML
• Activated in AIDS patients
• Fgr-myeloid cells, B cells
• Blk-B cells
• Lck- T cells, NK cells, brain, colon tumors (from
  an alternate start site)

6
Expression of Src family members

• Frk subfamily (Frk/Rak)/lyk/Bsk)-primarily in
  epithelial cells

All cells (probably) express multiple Src family
members
Multiple isoforms of the enzymes may exist In the
same cell
The kinases may function in multiple cellular
locations
7
Src Structure
adapted from V. Levin Cancer Treat Res
11989-119, 2004
8
Dummies Guide to Structure and Regulation of Src
Family PTKs
N-terminal phosphorylations
SH-4
416
527
SH-3
SH-2
SH-1
Y
Y
palmitate
Autophosphorylation site
myristate
Site whose phosphorylation by
Csk DECREASES Kinase activity
9
Structural domains of Src family members

• SH4
• 15 aa sequence that contains the signal for lipid
  modification
• Gly 2 is required for addition of myristic acid
  (all family members except Frk)
• Palmitoylation occurs on all members but Src
• Unique domain
• Distinct for each family member
• May facilitate interactions with specific
  receptors
• In Src and Lck, Ser/thr phosphorylations occur,
  usually during mitosis

10
Structural domains of Src family members(cont)

• SH3 (by defn) all contain proXXpro as a core
• 50aas,
• Regulates intra and intermolecular interactions
• Important in enzyme activity, cellular location
• Recruits substrates, especially
  cytoskeletal-associated proteins
• SH2 (all bind short, contiguous sequences
  containing p-tyr)
• Important role in regulating catalytic activity
• Localization, recruitment, binding partners

11
Structural domains of Src family members(cont)

• SH-1 (catalytic domain)
• Highly conserved, suggesting one primordial
  kinase gene
• Lacks sequence specificity in phosphorylation
• (if a protein can interact, and its got a
  tyrosine, its a likely substrate)
• C-terminal tail
• Lost in v-Src
• Contains a tyrosine phosphorylation of which
  inhibits kinase activity

12
Dummies Guide toRegulation of pp60 Protein
Tyrosine Kinase Activity
SH-2
SH3
Y
SH-1 (Kinase)
Intra-molecular interaction of Y-P with src SH-2
domain hides kinase domain
CSK (C-Src Kinase)
PTPase ?
13
CSK

• C-Src terminal Kinase
• Very sequence specific in phosphorylating the
  c-terminal region of Src family members
• Principal NEGATIVE regulator of Src activity
• Recruited to focal adhesions, where it may
  rapidly affect the c-terminal phosphorylation of
  Src
• Functional deletion in mice is lethal day 8-9
  when embryos are turning activity of Src
  family in Csk-/- cells is very high

14
Src Structure
From Xu et al Molec. Cell 3 629-638, 1999
Figure 1. The Assembled Conformation of c-Src.
Schematic illustrations of the human c-Src
structures reported here and the previously
determined human c-Src structure. In the Src-1
structure ( b), the activation loop is
disordered. In the structures of Src-2 to Src-5
(a), the activation loop forms an inhibitory
helix. To accommodate this helix, the cleft
between the two kinase lobes is about 5 more
open than in the Src-1 structure.
15
Src Structure
From Xu et al Molec. Cell 3 629-638, 1999
Figure 3. Ribbon Diagram Showing the Overall
Organization of Src-2The SH3 (yellow) and SH2
(green) domains coordinate the linker and
phosphorylated tail segments, respectively. The
tyrosine kinase domain is colored blue AMPPNP
(red) is bound in the active site. The A loop
helix packs between the N and C lobes of the
kinase and sequesters Tyr-416.
16
Comparison of Src and Lck
From Xu et al Molec. Cell 3 629-638, 1999
17
Src and Abl-Most Src Inhibitors are Abl Inhibitors
from Harrison Cell12 737-740, 2003
Figure 1. The Order of Domains in the Polypeptide
Chains of Src and Abl, and Diagrams of Their
Assembled, Autoinhibited States. In both cases,
the SH3-SH2 clamp fixes the bilobed kinase domain
in an inactive conformation. The domain color
codes are SH3, yellow SH2, green kinase small
lobe, dark blue kinase large lobe, light blue.
The activation loop in the large lobe is red.
Connector, linker, and N- and C-terminal
extensions are black. In Bcr/Abl, gene fusion has
replaced the Abl cap by a long segment of Bcr.
18
Src and Abl activation
Figure 2. Modes of Activation for Src and Abl
Unlatching, Unclamping, and Switching. In
Src-family members, the assembled state is
unlatched by dissociation of the C-terminal tail
from the SH2 domain and dephosphorylation of the
exposed Tyr 527.
from Harrison Cell12 737-740, 2003
19
Src domains and Src activity
Basal Activty (discerned from crystal structure)
Mechanisms of Activation
Activated Kinase
SH4
SH3 ligand binding
SH3
Recruitment of proteins
SH1
SH2
Y
phosphorylation
416
P
Y 527
SH2 ligand binding
dephosphorylation
Phosphorylation of substrates
Modified from Taylor and Brugge Ann. Rev. Cell
Dev Biol 13 513-609, 97
20
Functions of Src, Yes, and Fyn

• Required for proliferation from most, but not all
  growth factors
• Associates with most, but not all growth factor
  receptors
• Required for Cell Cycle Progression
• G1 transit
• M Phase
• Knock-outs of individual members are viable
• Required for migration
• Required for response from several types of
  stress (e.g. hypoxia)
• Involved in apoptotic pathways
• Involved in angiogenic pathways
• Alternate-splice forms participate in neural cell
  function

21
Functional deletion of Src family members

• Src-/- mice are viable
• Mice develop osteopetrosis, a defect in bone
  remodeling
• Src expression is very high in normal osteoclasts
• Src-/- mice are reduced in tyrosine
  phosphorylation
• The osteopetrosis defect can be overcome by
  expression of a kinase inactive Src, suggesting
  another function (scaffolding?) for Src in
  osteoclasts

22
Functional deletion of Src family members (cont)

• Yes KOs have no obvious defect
• Fyn KOs are viable, but may have reduced
  response through T cell receptors (TCR)
• Also abnormalities in brain development
• Lck KOs are viable
• Significantly reduced in CD4/CD8 lymphocytes
• Severe defects in TCR signaling by antigens
• Lck thus plays a prominent role in T cell
  development

23
Redundancy in Src family functions(as suggested
from knock-out studies)

• Lck and Fyn play overlapping and somewhat
  distinct functions in T cell development
• Lck-/- Fyn-/- mice have more severe T cell
  defects
• Src-/- Hck-/- mice suffer much more severely from
  bone defects
• Src-/- Fyn-/- mice die perinatally
• Src-/- Yes-/- MAY be viable
• TAKE HOME MESSAGE-Src family members PROBABLY
  play overlapping, not strictly redundant
  functions

24
Transgenics and KOs in tumorigenesis

• Polyoma Middle T (mT)
• Transforming protein of the DNA tumor virus,
  polyoma
• Transformation REQUIRES association with a Src
  family member (Src, Yes or Fyn)
• Transgenic mice expressing mT driven from an MMTV
  promoter develop mammary carcinoma

25
? Which Src family member is required

• EXPERIMENT
• Cross mT mice with src-/- yes-/- fyn -/-
• Assess tumorigenicity
• RESULTS
• In a Yes -/- or Fyn-/- background, tumors develop
• In a Src-/- background, tumors DO NOT develop
  (mostly)
• CONCLUSIONS
• Activated Src contributes to tumorigenicity
• Mutations are not required
• These conclusions seem to hold for many human
  tumors

26
The World According to Src
RPTKs
mitosis
G protein coupled receptors
antigens
cytokines
cytoskeletal reorganization
Src Family
oxidative stress
mitotic functions
extracellular matrix
stress pathways
PI-3-kinase
myc
ras
angiogenesis
27
Act I Receptor Protein Tyrosine Kinases
HGF
PDGF
VEGF
V. Levin, Cancer Treat. Res.
28
Act II Extracellular Matrix
V. Levin, in Press
29
Act III Motility and Invasion
Fibroblasts from src-/- mice are impaired
in motility
V. Levin, in Press
30
Act IV Disruption of Endothelial Barriers
Src inhibition blocks tumor cell Extravasation
Weis et al., 2004
VE
endotheial barrier disruption
31
Act V Chemoresistance
Src Expression/Activity
Increased Src Expression/Activity
Drug Resistance
32
Act VI Roles of Src in Angiogenesis
SRC
Regulation of Angiogenic Molecules Produced by
Tumor Cells
Regulation of Endothelial Cell Function
33
The Final Act (for now) Src in the Clinic
gt20 trials are underway What will they
target? Who will benefit?
NOW-about 90 trials are in progress
34
Src Inhibitors in the Clinic2005-
35
2005-0842 Phase IB study of Dasatinib,
Cetuximab, and FOLFOX in patients with
metastatic adenocarcinoma of the colon or
rectum. Patient 01 Previously refractory to
FOLFOX and Cetuximab
Baseline
After 3 weeks of treatment
Scott Kopetz, M.D.
36
A src Chauvinists View of Signal Transduction
polypeptide growth factor
Extracellular matrix integrins fibronectin
EGF-r Her2/neu CSF-1 etc.
Hypoxia
fak
SRC
vEGF
cdc-2
NUCLEUS
stat-3
Epithelial and fibroblast
37
Association of Src with receptors

• Src family members associate with T cell and B
  cell receptors and are functionally important
• Src family members associate with receptor
  tyrosine kinases (PDGF-R, FGF-R. TrkA, EGF-R,
  c-Met, c-Kit, Insulin and Insulin like receptors,
  etc.)
• Src family members participate in signaling
  complexes mediated by integrins and other
  adhesion receptors
• Src family members are frequently activated by G
  protein-coupled receptors (e.g. LPA, thrombin,
  angiotensin II, bradykinin, vasopressin and many
  others) role of Src family in these processes
  and mechanism of activation is not well described
• Src family members associate with cytokine
  receptors
• Src family members associate with GPI-linked
  receptors (uPAR, CD14,24, Thy-1 and others
• Src associate with (or respond to activation of)
  classic hormone receptors

38
Association of Src with Growth factor receptors
EGF
PDGF
SH4
EGF-R
SH4
Neu
Y
P
PI3K
Y
PDGF-R
Y
Src associates with HER1/HER2 Dimers
phosphorylates HER1 (EGF-R)
PDGF-R induces N-terminal Src phosphorylation
Direct association of Src family via SH-2
interaction Evidence this phosphorylation site
might predict response to some EGF-R inhibitors
39
PI3K activity
Fatty acids
phosphatidic acid
O
O
O
O
glycerol
PLC
2
1
3
P
OH
ATP
2
OH
6
1
OH
PI3K
4
OH
OH
3
5
P
ADP
Modified from Vanhaesebroeck And waterfield Exp
Cell res, 1999
40
Akt/PKB-mediated signals
PH
T308
S473
reg
Catalytic
GSK3
PFK-2
PDE-3B
mTOR
IkB
Bad
Casp-9
eNOS
Forkhead TX Factors
Glycogen synthesis
Protein synth
glycolysis
cAMP
translation
Exp Of antiapoptotic genes
Bcl-XL Bcl-2
Expression of Fas ligand
Modified from Vanhaesebroeck and Waterfield Exp
Cell res, 1999
SEE PREVIOUS LECTURE
41
Akt activation
PH
T308
S473
Growth factor/ PTK receptor
Integrins/FAK
reg
Catalytic
Akt/PKB
PI3-Kinase
PI(4,5)P2
PI(3,4,5)P3
MMAC/PTEN
PDK1
SHIP
PDK2 ? ILK ? PDK1 ?
Tumor suppressor gene discovered At MDACC
PI(3,4,)P2
42
Association of Src with Growth factor receptors
PTPase
Uncoupling Src with HER-2/neu leads to Inhibition
ofPTEN function (D. Yu)
Regulation is a two-way street
43
Association of Src with Growth factor
receptors(Cont.)
Insulin Receptor
b
Indirect association through Insulin receptor
Substrate (IRS)
44
Src Mediates Receptor Tyrosine Kinase Function
Bromman et al., Oncogene 48 7957-68, 2004.
Figure 2 A model for how SFKs participate in RTK
signaling. Oncogenic forms of SFKs phosphorylate
RTKs. SFKs activated by cellular signaling also
phosphorylate RTKs (in the example shown, SFKs
are activated by a G-protein-coupled receptor
(GPCR)). Once activated by RTKs, SFKs regulate
receptor turnover both at the level of
endocytosis and ubiquitination. SFKs also
participate in RTK-stimulated cytoskeletal
reorganization, migration, and survival
45
Association of Src with receptors-Summary

• Association may be direct or indirect
• Src activity is increased by multiple mechanisms
• Receptor activity may (or may not) also be
  affected
• Src is likely to mediate cross-talk between
  receptors
• Src family activation may be required for some
  receptor functions, including proliferation
• Src family activation may be redundant in some
  functions (e.g. most RPTKs and Src can BOTH bind
  and activate PI-3 kinase

46
Src regulation of GPCRs
From Luttrell and Luttrell. Oncogene 23
7969-7978, 2004.
SH-3-mediated
Figure 1 Src family kinases as direct effectors
of GPCR signaling. Hormone (H) binding to a GPCR
catalyses GTP for GDP exchange on heterotrimeric
G-protein G subunits, G-protein dissociation, and
activation of effectors (E) by free G-GTP and G
subunits. Desensitization of activated receptors
involves phosphorylation by GRKs, which promotes
-arrestin (-arr) binding. GPCR-bound -arrestins
also bind to clathrin and the 2-adaptin (AP-2)
subunit, leading to receptor clustering in
clathrin-coated pits and dynamin (dyn)-dependent
endocytosis. Recruitment and activation of Src
family kinases, caused by binding to
proline-rich motifs in the intracellular domains
of certain GPCRs or interaction with G subunits,
have been described. In addition, Src family
kinases can bind to arrestins, which confers
tyrosine kinase activity upon a complex
assembled on the desensitized GPCR
47
Review of Last LectureNuts and Bolts of Src
Basal Activty (discerned from crystal structure)
closed conformation Basal (low) activity
48
Src mediates crosstalk between GPCRs and Focal
Adhesions
From Luttrell and Luttrell. Oncogene 23
7969-7978, 2004.
Figure 3 Activation of Src family kinases through
crosstalk between GPCRs and focal adhesions.
GPCR-mediated activation of phospholipase C
(PLC) increases intracellular calcium (Ca2i) and
activates PKC. In neuronal and hematopoietic
cells, Pyk2, an FAK family nonreceptor tyrosine
kinase, is activated in response to the rise in
intracellular calcium and PKC activity. Pyk2,
like p125FAK, binds and activates Src, leading to
the assembly of a focal adhesion- based complex
containing a number of signaling proteins, such
as paxillin, CSK, and the Crk/C3G and Grb2/mSos
Ras-GEFs. Recruitment of Ras-GEF activity
promotes Ras activation and Ras-dependent
stimulation of the cRaf-1, MEK1/2, and ERK1/2
MAP kinase cascade. This form of GPCR-stimulated
Src activation is conditional, in that both a
calcium/PKC signal and the adhesion-dependent
integrity of focal adhesions are necessary for
signaling
49
Integrin-mediated Src activation

• Following engagement of integrins with their
  ligands (e.g. fibronectin, etc., etc.,
  etc.,integrins transduce signals involved in
  adhesion, cell spreading, migration,
  proliferation, differentiation and other minor
  events
• Integrin aggregation increases tyrosine
  phosphorylation in cells, and activates Src and
  Focal Adhesion Kinase (FAK)
• Src associates with several proteins in focal
  adhesion complexes including paxillin, tensin,
  vinculin, Cas, FAK, PI3-K, Csk and others
• Src phosphorylates FAK in the complexes,
  increasing FAK activity and inducing further
  recruitment of proteins
• Thus, Src acts both as a kinase and in formation
  of complexes

50
Focal Adhesion Kinase (FAK)

• First discovered as a substrate for v-Src
• Now, known to be a critical mediator of integrin
  signaling
• Important in the formation and turnover of focal
  adhesions, critical to cell migration
• Role in proliferation is controversial
• Mediator of a cell survival pathway

51
FAK structure (Dr. Sood)
Crk
Csk
Nck
Cas
Integrin
talin
Crk
Sos
paxillin
FAT Region
Amino Terminal Domain
Kinase Domain
Carboxy-terminal Domain
Y397
Y925
Grb-2
Src
PI3-K
Sos
FAT Region Focal Adhesion Targeting Region
52
Src and Migration
From Playford and Schaller, Oncogene 18
7938-46, 2004
53
Playford and Schaller, Oncogene 18 7938-46, 2004
54
Requirement of Src for growth factor-induced DNA
synthesis
Ras
Mapk
?
Ets
Fos/Jun
55
Requirement of Src for growth factor-induced DNA
synthesis
PDGF
Experiment Transfect a dominant negative Src
family construct into cells Result DNA
synthesis induced by PDGF, EGF,Csf-1 is
abrogated Conclusion Src activation IS
required for many growth factors to induce DNA
synthesis
PDGF-R
SH4
Y
P
Y
Ras
Mapk
?
What is Src required For
myc induction
Ets
Fos/Jun
56
Src and mitogenesiss
Bromman et al., Oncogene 48 7957-68, 2004.
Figure 1 A model for how SFKs mediate mitogenesis
in response to RTK activation. RTK-activated
SFKs positively regulate DNA synthesis via
stabilization of myc mRNA and thus increase the
production of Myc, a transcription factor
required for mitogenesis. Although perhaps
dispensable for stabilization of myc
mRNA, RTK-dependent activation of the traditional
Ras/MAPK pathway stabilizes Myc protein in
response to growth factor stimulation. The
mechanism of SFK-induced myc mRNA stabilization
is unknown, but might involve the activation of a
number of effectors including Abl, Shc, Stat3,
Vav2, and Rac, as these are required
for SFK-mediated DNA synthesis in response to
mitogens (see text, for details). SFKs also
stimulate DNA synthesis by opposing the negative
growth effects of p53 and PKC, although whether
either of these effectors is involved in
SFK-mediated myc mRNA stabilization is unknown.
Finally, SFK activation results in translocation
of RasGRP1 to the Golgi network and activation of
Ras signaling there. Whether Ras signaling within
the Golgi participates in mitogenesis is not
clear
57
Src and Cell cycle ProgressionG2/M transition

• V-Src accelerates meiotic maturation in Xenopus
• In somatic cells, c-Src activity is increased
  during mitosis
• Experimentally, how would you tell?
• Src is phosphorylated at serine and threonine
  residues by Cdc2 kinase
• Src associates with Cdc2 and Sam 68, involved in
  RNA processing
• G1?
• Src is required here as well
• Inhibitory Src antibodies block cell division if
  injected throughout G1
• Conclusion Src family plays a critical function
  required for progression from G2 to cell division

58
Src Activation in Human Tumors

• No mutated enzymes have been found (probably)
• Never found in transfections
• High Specific Activity Occurs in
• Colon Cancer
• Prostate Cancer
• Pancreatic Cancer
• Gastric Cancer
• Breast Cancer
• RELATED TO HER-2/neu?
• WHAT ARE THE MECHANISMS?

59
Src-mediated Signaling Pathways
Summy and Gallick, CCR 2006
60
Src and Regulation of Gap Junction Communication
in Transformed Cells
Src orchestrates cross talk between pathways
that regulate gap junctional communication. Src
can activate or interact with other kinases by a
variety of methods. Kinases that increase or
decrease communication are indicated in green and
red, respectively
Pahujaa et al.Exp Cell Res.3134083-90, 2007
61
Src and Regulation of Gap Junction Communication
in Transformed Cells
Pahujaa et al.Exp Cell Res.3134083-90, 2007
62
Src and Regulation of Gap Junction Communication
in Transformed Cells

• Phosphorylation sites on Cx43. (a) Monomeric Cx43
  with phosphorylation sites targeted by known
  kinases are labeled by amino acid number and
  phosphorylating kinase. (b) Side view of a Cx43
  gap junction channel. (c) View from inside of a
  cell looking towards an adjacent cell. Events
  that increase or decrease communication are
  indicated in green and red, respectively.
  Established phosphorylation sites conserved
  between humans, rodents, primates, and pigs are
  shown. Cysteines forming disulfide bridges in the
  extracellular loops are shown in purple.
  Transmembrane helices are in dark green with
  phospholipid heads on the plasma membrane shown
  in orange. This illustrative model of monomeric
  Cx43 was assembled using WebLab ViewerPro 4.0
  (Accelrys). Four a helices (dark green) were
  aligned with a previously reported model of the
  transmembrane portion of Cx32 (PDB code 1txh)
  66. The extracellular ends of the helices were
  connected (M1 to M2 and M3 to M4) via two ß
  hairpins stabilized by three cystine bridges
  (purple) each as suggested by Sosinsky and
  Nicholson 1. The cytosolic end of the M4 a
  helix was connected to a C-terminal tail
  structure of rat Cx43 (PDB code 1r5s) 68.
  Finally, random-coiled protein fragments of
  appropriate lengths were used as the N-terminal
  portion of Cx43 and as the intracellular loop
  connecting helices M2 and M3. Monomers were
  assembled into hemichannels by aligning them with
  the model described by Fleishman et al. 66 (PDB
  code 1txh). Hexamers were imbedded into a model
  of fluid lipid bilayer as described 80.
  Hexamers were then assembled into gap junctions.
  It should be stressed that the cytoplasmic tail
  and intracellular loop are flexible and may
  assume different structures 68. These
  hypothetical models are simply meant to
  illustrate potential special differences between
  positive and negative regulatory phosphorylation
  sites with reference to the channel structure.

63
How Does Src Function in Tumorigenesis/Progression

• Probably has little affect on proliferation
• Strongly promotes expression of pro-angiogenic
  factors
• Clearly Effects Migration/Invasion and a process
  called Epidermal/Mesenchymal Transition (EMT)

64
Src, Angiogenesis and other Survival Pathways

• Src upregulates one critical mediator of
  angiogenesis, vascular endothelial growth factor
  (VEGF)
• Under conditions of hypoxia, Src activation is
  required as Src-/- fibroblasts are severely
  impaired in hypoxic induction of VEGF
• Reduction of Src (previous slide) in colon tumor
  cells results in reduced VEGF expression
• Src also regulates PI-3K, Akt mediated
  anti-apoptotic pathways the direct connection
  remains to be elucidated
• See Schlessinger J. New roles for Src kinases in
  control of cell survival and angiogenesis.
  Review Cell. 100(3)293-6, 2000.

65
STAT Signaling
From Silva. Oncogene 238017-23, 2004.
66
Src, STAT3 and Tumorigenesis
From Luttrell and Luttrell. Oncogene 23
7969-7978, 2004.
Bclxl
MMP-2
VEGF
67
Regulation of Hif-1 Binding to the VEGF Promoter
Normoxia
VHL
VHL regulates HIF1a degradation Activated
proto-oncogene pathways Increase VEGF translation
VEGF Promoter
HIF-BE
STAT3-BE
HIF-BE
68
Src Regulation of VEGF Transcription Through STAT3
Normal Cells
69
Src Mediates Pathways Leading to Expression of
IL-8 and VEGF
NFkB
IL-8
70
Transformation by v-Src
71
Src, Migration, Invasion and Promotion of
Mesenchymal-like Phenotype
Growth factor receptors
Integrins
E cadherin
Src
Src
Fak
E cadherin
Src
Src
Src
Src
Src
Src
Src
Src
Normal Epethelium
Tumor Progression/drug-resistance?
Eidermal-to Mesenchymal-Like transition (EMLT or
EMT)
P
P-myosin vimentin
P
P
Src
Src
Fak
Src
Fak
Src
Rac
Erk1/2
MLCK
Mesenchymal-like Phenotype Loss of Cell-Cell
Adhesion Increased Migration
72
Morphological differences in gemcitabine
resistant pancreatic adenocarcinoma cells
Gemcitabine resistant Cells
L3.6pl Parental
Ami Shah, M.D.
73
PREDICTION

• Src inhibitors, functioning in angiogenesis,
  migration, invasion will have primary effects not
  on tumor growth, but on metastasis

74
The Classic Autocrine Regulatory Loop
tumor cell
EGF-R
Normal regulation of colonic epithelial
cells Normal response to wounds
Signal Transduction is Signal Transduction, Not
Biologic Context
75
Autocrine Regulation by VEGF in Tumor Cells?
tumor cell
TGFa HGF
EGF-R C-Met
Increased tumor cell migration Increased VEGF
expression
In collaboration with Lee Ellis et al.
76
Roles of Src and VEGF in Endothelial Cells and
Tumor Cells
(SEE Weis and Cheresh, Nature 437 497-504,
2005)
Will Src inhibitors Have greatest Efficacy
for Metastasis-inhibition?
VEGF-R
endothelial cell
Increased EC permeability Increased migration
Increased tumor cell migration Increased VEGF
expression
Spontaneous metastasis is impaired in Src-/- mice
77
Src Activation in Tumor and Host CellsSrc Cycle?
Implication Src Activity in Tumor Cells Effects
Src Activity in Endothelial Cells
78
Predictions

• Src ITSELF will have minimal effects on tumor
  growth-borne out in Src-/- mouse studies
• Usefulness of Src inhibitors in inhibiting growth
  of primary tumors would depend on their ability
  to affect additional Src Family Kinases (SFKs)
• Src, Yes, or Fyn is required for proliferation
• Src inhibitors may have efficacy in inhibiting
  metastasis
• Src inhibitors are unlikely to affect tumor
  growth at most metastatic sites yet that is the
  focus of some trials

79
Effects of siRNA Silencing on Src Expression in
L3.6pl Pancreatic Adenocarcinoma Cells with High
Metastatic Potential in Orthotopic Mouse Models
siSrc Control (Empty Vector)
Wild-type Parental
SiSrc Clone 2
SiSrc Clone 1
c-Src
Trevino and Summy
80
Downregulation of Src does not affect L3.6pl
growth in Vitro
4.5
4.0
3.5
3.0
Absorbance (450 nM)
2.5
2.0
1.5
1.0
.5
0
24
0
12
36
48
Time, hrs
Trevino and Summy
81
 
Effects of Cell-dependent c-Src Targeted siRNA
In Vivo Growth and Progression of Pancreatic
Adenocarcinoma Cells
Incidence
 
  L3.6pl control cells or clonal variants stably
expressing c-Src targeted siRNA, 1.25, 2.5, and
5.0 x 105 cells/mouse, were injected into the
pancreas of nude mice on Day 0. After 6 weeks,
all mice were sacrificed and evaluated for
primary tumors and liver and lymph node
metastases.   Plt0.05, relative to controls
Trevino, Summy et al., AJP, 2006
82
Effects of c-Src Src Family Kinase Selective
Inhibitor Dasatinib on In Vivo Growth and
Progression of Pancreatic Adenocarcinoma Cells
 
Incidence
L3.6pl cells were injected into the pancreas of
nude mice (5 x 105 cells/mouse) on Day 0. On Day
14, 200 ?l dasatinib (15 mg/kg) or an equal
volume of citrate buffer vehicle was administered
by oral gavage. Treatments continued daily for
28 days. Mice were sacrificed on Day 42 and
evaluated for primary pancreatic tumors and liver
and lymph node metastases.   Plt0.05, relative
to controls
Trevino and Summy
83
Where are we now?

• Src inhibitors ARE showing success in the clinic
• Combinations with chemotherapy to overcome
  chemotherapy resistance
• Prostate Cancer where both Src and Lyn are
  activated
• International Phase III trial started 11/08
• Bone metastases where Src inhibitors block bone
  degradation

84
Where do we need to go

• Back to the lab
• Back to the clinic
• What are the mechanisms by which Src becomes
  activated in resistant cells?
• What are the mechanisms by which Src inhibitors
  reverse resistance?
• What tumor/Stromal Targets are critical for
  success

85
BUT

• Science ALWAYS is full of surprises

86
Like
Stress Hormones, e.g. Norepinepherine
Midterm Exam
Armaiz-Pena and Sood, unpublished
87
Src Structure
mutate serine to alanine