Involvement of Angiogenic Factor in Cancer and Inflammation

------- Focusing on VEGF System -------

2007.3.9

Masabumi Shibuya

‡：The figures, photos and moving images with ‡marks attached belong to their copyright holders. Reusing or reproducing them is prohibited unless permission is obtained directly from such copyright holders.

１.Fujinami Sarcoma Virus --------------- v-fps cancer gene 1913 Prof.Hanafusa 1980 Shibuya et al. Cell,1982

２. Discovery of structural anomaly in c-ErbB (EGFR)gene

in human brain tumor (glioblastoma)

gag-v-Fps

FSV DNA

gag

v-fps

SH2 tyrosine kinase

EGF receptor

deletion mutation

EGF non-independent activation

Yamazaki et al. Mol Cell Biol,1988; Yamazaki et al Jpn.J.Cancer Res. 1990.

VEGF Independent/Non-Independent Vascular System Regulation

１. Characteristics of VEGF system and comparison with other angiogenic factors. Characteristics of the receptor VEGFR2.

２. Responsibility of VEGFR1 to metastatic carcinoma, inflammatory diseases, and pregnant toxicosis

３. Toward regeneration of vessel, characteristics of VEGF-E, mechanism of vascular permeability

４. Regulation of VEGF non-independence

５. Summary of angiogenesis inhibiting treatment

Blood Vessel and Lymph Vessel of Skin

width 1 mm ‡

VEGF basic FGF etc.

Vascular Endothelial Cells involved in Cancer1. Tumor angiogenesis 2. Abnormal vascular permeability 3. Metastasis 4. Malignant transformation of endothelial cells.

Angiogenic factors

Tumor cells

Tumor Angiogenesis

Tumor growth Metastasis Ascites

Malignant Conversion of Cancer and Vessel：Propagation, Transition, Ascites

cancer cell

angiogenic factors

tumor vessel, transition

１. formation of tumor vessel ２. abnormal enhancement of vessel permeability ３. hematogenous metastasis ４. canceration of epithelial cell

neovessel・spread of lymph nodes

VEGF-VEGF Receptor System

cleavage

VEGFR-1 VEGFR-2 VEGFR-3

sVEGFR-1

(Flt-1) (KDR/Flk-1) (Flt-4)

Nrp-1

PlGF VEGF-B VEGF-E VEGF-D

VEGF-C

Birds: 3VEGFR system Drosophila: 1VEGFR system

svVEGF VEGF-A

neogenseis of lymph vessels

Nrp-2

Genesis/neogeneis of blood vessels

Interaction between angiogenic regulation system except VEGF

membrane surface of epithelial cell

Fish

Frog

Reptile

Birds

Mammals

Vertebrates

Non-vertebrates

Drosophila 1 VEGFR

3 VEGFRs with soluble VEGFR1

4 VEGFRs

Phylogeny of 7 Ig-tyrosine Kinase Receptor (VEGFR family）

Receptor-type Tyrosine Kinases

Growth stimulation Angiogenesis Vascular permeability autocrine

paracrine constitutive paracrine

VEGF (PlGF)

VEGFR

VEGFRs (Flt-1, KDR/Flk-1)

Cancer Cell

Endothelial Cell

αEGFTGFFGFetc.

EGFR etc.

and Carcinogenesis

Role of Tyrosine Kinase in Canceration

Paracrine Effect of VEGF and Its Receptor

host’s mesenchymal cell

Tyrosine Kinase Receptors and Intracellular Signaling

MAPK

Raf1

JAK

STAT PKC

Shc Sos Ras PI3K

PLCγ Grb2

MEK

Rac Rho

Akt

Nucleus

EGFR NIH3T3: +++ +++ VEGFR-1 NIH3T3: - -/(+) VEGFR-2 NIH3T3: - +

Transform DNA synth with ligand

VEGFR-2 (KDR/Flk-1)

Characteristics of VEGFR-2 signal transduction： mainly uses C-kinase system, not so dependent on Ras system

+/+ Flk-11173F/1173F Flk-1KO/KO

E8.5

E9.5

PECAM-1 immunostaining

PECAM-1-Positive Blood Vessels Were Absent in Flk-11173F/1173F and Flk-1KO/KO Embryos

ys, yolk sac

ys ys

ys

ys ys

ys

Sakurai et al. PNAS, 2005

‡

HIFα

basic amino acids

spacer

New bipartite NLS Nuclear transport

VEGF

VEGFR-2 (KDR)

p PKC

PLCγ

D N A s y n t h e s i s A n t i b o d y

Vascular endothelial cell

N U C L E U S Tumor cell, astrocyte, pericyte ---.

Hypoxia or VHL(-) condition

Y-1175

Y-1214 P PI3K-Akt

Survival ?

cf. Zebrafish mutants with angiogenesis defect. cf. PDGFR, D-PVR

DNA synthesis

basic amino acids

MAPK

Induction of VEGF Expression and Cell Proliferative Signal from VEGF Receptor-2 use pY1175 - PLCγ - PKC - MAPK for DNA synthesis

Enhancing Activity of VEGF on Vessel Permeability --- Miles Assay

ascites Mouse ascites cancer cell MM2

(8 days)

right： control

left：anti-VEGF neutralizing antibody administrated

PBS

PBS

In every mouse ascites cancer, large amount of VEGF is accumulated in ascites.

‡

‡

Models for Ascites Formation

1. other VPFs (Bradykinin, Histamin etc.) 2. Suppression of absorption by cytokines 3. Abnormal coagulation

Carcinoma & Sarcoma

Lymphatic vessels Blood vessels

normal VEGF

Lymphoma & Leukemia

abdominal wall

abdominal cavity

Block of lymphatic vessels

Local high conc. of VEGF

(1)

(2)

VEGF

Ascites

Ascites

VEGF

Sensitive to Neutralizing Antibody

Resistant to Neutr. Ab ?

Mouse ascites cancer

MM2 breast cancer-derived cell strain.

VEGF: 85 ng/ml ascites

anti-mouse VEGF antibody therapy (1w)

decrease in ascites ( to 35%)

decrease in tumor cells ( to 35%)

decrease in bleeding ( to 10%)

VEGF+/- VEGF+/+

?

normal

?

lethal vascular defect

lethal over growth

Survived Macrophage: VEGF-dep.migration (-)

(Flt-1) (Flk-1)

Flt-1 TK-/-

Flt-1 TK -/- Flt-1 -/-

Knockout Mouse Analysis of VEGF, VEGF Receptor Gene

VEGFR1 VEGFR2

Tumor W TK(-) W TK(-) W TK(-)

Tumor

W, wild type; TK(-), VEGFR-1 TK(-/-); Tumor: Lewis lung ca

Tumor Lung meta

Mouse Model of Tumor Transition to Lung

Transition to lung decreases in a mouse with VEGFR-1(Flt-1) signal defect.

‡ ‡

Pulmonary induction of MMP9 via primary tumor is significantly decreased in VEGFR1 (flt-1) TK(-/-) mice.

MMP9 MMP2

W W L H H L W W B H H B lung

W: wild type; H: VEGFR-1 (flt-1) TK-/-

L: Lewis lung carcinoma; B: B16 melanoma

liver

kidney

spleen ‡

Close Relationship between Flt-1 and MMP in Lung Tissue.

lung

Tumor

Tumor tissue

(x)

VEGFR-1(Flt-1)

MMP9 induction Metastasis

Lung endothelial cell macrophage 　　

VEGFR-1 TK(-/-) and MMP9(-/-) block this phenotype.

TK(-/-)

MMP9(-/-)

Cancer Cell. 2003

(c.f. Nature 2005)

Mechanism of New Cancer Transition Dependent on VEGF Receptor-1 (Flt-1) Kinase

Expression of Soluble VEGFR1 (flt-1) mRNA in Placenta

In pregnancy toxemia, sol. Flt-1 in mother’s blood increases abnormally.

Full length

Soluble form

pregnancy toxemia

normal pregnancy

age in month ‡

New Type Angiogenic Factor, VEGF-E

cleavage

Binds only to VEGFR-2 Promotes angionenesis well in vivo. Epithelial cells adhere well. edema（ー） hemorrhagic lesion（ー）

Cell membrane

control

Phenotypes of Human VEGF-A165 Transgenic mouse

VEGF-A165 Tg control VEGF-A165 Tg

VEGF-A165 +++ (thin) +++ +++ +++ (2nd, +) VEGF-E (NZ7) +++ + - -

blood vessels leakiness inflamm. body w. loss

PlGF + - - -

‡ ‡

When VEGFR1 and VEGFR2 are activated at same time, promoting signal of vascular permeability increases.

R1 R2 R1 R2

VEGF-A165 PlGF VEGF-E T.f. svVEGF

VEGFR1 (Flt-1)

VEGFR2 (KDR/Flk-1)

R1 R2

VEGF activity

VPF activity

+++ +/- +++ +

+++ + + +++ (++ in acute phase)

Biological Functions of VEGFR-1 (Flt-1) in Mammal

Inhibitory regulation of angiogenesis during fetal life

Barrier function in placenta? (pregnancy toxemia)

(1)

(2)

Ambulato of macrophage

(3)

Slight angiogenesis, high promoting of permeability

(4)

Reconstruction of bone marrow

(5)

MMP9 induction, promotion of transition to lung, promotion of inflammation

(6) Expressing cells:

vascular endothelial cell, macrophage, haematopoietic stem cell , plain muscle cell

Summation of Angiogenesis

Pro-angiogenic Anti-angiogenic

Capillary Network in Rat Pupillary Membrane at Day-8.

Lens epithelial cell produces BMP4, and induce apotosis of blood vessel.

‡ ‡

Rat Pupillary Membrane with Vascular Network

Day-8 rat

PM Lens Cornea

B c l X L B c l X s

L C P M L C P M Day-8 One day culture BMP4

‡

‡ ‡

‡

surgery actinotherapy

chemotherapy

anti-angiogenic therapy

molecular targeting

immunotherapy

VEGF (VEGF-A) Neutralizing Antibody

the third phase clinical experiment Stage-3,-4 colon cancer patients: randomized trial (400 x 2)

chemotherapy ± anti-VEGF antibody

survival time：ーantibody, 15.6M; + antibody, 20.3M

by-effect：slight high blood pressure（no bleeding）

strain on kidney ーantibody + antibody

cf. surgical benefit in breast cancer and lung cancer (NSCLC)

N. Ferrara et al. (Genentech Inc.), Gordon Conf. 2003, etc.

Laboratory to (Bench) Clinic (Bedside) ----- Cancer Therapy by Tumor Vessel Inhibition----（１） Why Drug Discovery Was Efficient in VEGF-VEGFR System 1. VEGF-VEGFR system was almost the central system of

vascular regulation in mammals. 2. Fundamental research in lab and application research in

firm supported each other well. 3. Similar effect of sol-Flt-1(s-VEGFR1) was recognized

without anti-VEGF antibody. 4. Cancer patients participated positively, which enabled steady

clinical experiments. 5. By-effects not being so heavy could be predicted not only

from animal experiments, but from the case of pregnancy toxemia.

Laboratory to (Bench) Clinic (Bedside) ----- Cancer Therapy by Tumor Vessel Inhibition---- （２） Challenge That Lies Ahead in Japan and the World 1. Clinical experiments on VEGF~VEGFR inhibitor must be proceeded

immediately in Japan and Asian countries. 2. Is there any difference in the effect between races? How about in stomach

cancer or liver cancer? Any effect on scirrhous gastric cancer? 3. Are there any patients resistant to anti-VEGF antibody? Is tumor vascular

inhibitor needed to be administrated individually? Are there individual variations in bi-effects?

4. What happens and what should be done when effects of anti-VEGF antibody

lower? Any other inhibitor of VEGF-VEGFR system (TK-inhibitor etc.)？ Any dependence on systems other than VEGF?---- FGF, PDGF etc. Any non-vascular-dependent propagation of cancer? New evaluation system?

To Young Researchers ----From My Experience-------

１. Find a rule used individually.

------- cyclic GMP

２. Concentrate when needed.--- From cancer gene to blood vessel.

３. Basically, research is a personal duty, but it cannot be done without supports from many people.

Experience in TR and clinics in Japan, proactive participation of patients is essential.

４. Acquire a global sense------ Lesson from studying abroad（Hanabusa Lab）

VEGFR-1(Flt-1) Shimehito Matsu Sachiko Yamaguchi Asako Sawano Toshio Ikeda Akira Yamane Kenji Wakitani Keiji Tanaka Sachie Hiratuka Yoshiro Maru Shinobu Iwai Kunio Kondo Lata Seetharam Satuki Kobayashi Masato Murakami Rei Koide Makoto Watanabe Tsuyoshi Nagase Kumi Iwata Akira Muramatsu

Acknowledgements：Co-Researchers

VEGFR-2(KDR/Flk-1) Tomoko Takahashi Yoshiko Sakurai Naoyuki Yabana Kaori Oogimoto Ako Masuda Junko Kami Takahiro Kamiga Saeed Samarghandian VEGF/VEGF-E/svVEGF /BMP Jin-Cai Luo Sachiyo Ogawa Atsushi Kiba Mari Seino Momomi Saito Hiroyuki Takahashi Yujuan Zheng Mai Yamauchi Seiji Yamamoto

External institutions, labs Tetsuo Noda（Tohoku U.） Nobuaki Yoshida（I. Med. Sci., U. Tokyo ） Shunpei Niida（NCGG） Tatsutoshi Nakahata（Kyoto U.） Shinichi Nishikawa（Riken） Kanji Sato（Tokyo Women’s Medical U.） Hitoshi Yoshizawa（Nara Medical U.） Mayumi Ono（Kyushu U.） Kensuke Egashira（Kyushu U.） Kari Alitalo (Helsinki U.) Peter Carmeliet (Belgium) J. Waltenberger (Germany) Kyowa Hakko Laboratory （Kenya Shitara etc.） Kirin Beer （Kazuhide Nakamura etc.） Daiichi Seiyaku（Noriko Tanaka etc.）

Cancer gene・tyrosine kinase Noriko Goto Lu-Hai Wang Jun Yokota Hitoshi Yamazaki Kiyoshi Ariizumi Toshihiko Seki Yoshiyasu Kaneko Shinsaku Hirosawa 林 雪芬 Misako Sato Bertrand Pain

Acknowledgements：Co-researchers（２）

Prof. Yoshihito Kamishiro, Prof. Takashi Sugimura, Prof. Haruo Kanno Prof. Hidesaburo Hanabusa, Prof. Kumao Toyoshima, Prof. Takeshi Odaka

External institutions, labs Yoshihito Kamiyama（Tokai U.） Kenichi Tamaki（Tokai U.） Shigero Mori （I. Med. Sci., U. Tokyo ）

Arinobu Tojo Yasuhisa Fukui Terumasa Tsuchiya Atsushi Kanno Sachiko Misawa Masayuki Hino Katsuya Yamamoto Michiko Kido Masashi Toyoda Taku Watanabe